Methods and devices for high fidelity polynucleotide synthesis

ABSTRACT

Disclosed are methods for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides. In exemplary embodiments, the methods involve synthesis and/or amplification of different oligonucleotides immobilized on a solid support, release of synthesized/amplified oligonucleotides in solution to form droplets, recognition and removal of error-containing oligonucleotides, moving or combining two droplets to allow hybridization and/or ligation between two different oligonucleotides, and further chain extension reaction following hybridization and/or ligation to hierarchically generate desired length of polynucleotide products.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/060,178, filed Mar. 29, 2011, which is a national stage applicationof International Application No. PCT/US2009/055267, filed Aug. 27, 2009,which claims the benefit of and priority to U.S. Provisional ApplicationSer. No. 61/092,309, filed Aug. 27, 2008, and claims the benefit of andpriority to U.S. Provisional Application Ser. No. 61/235,677, filed Aug.20, 2009, the entire contents of which applications are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

Methods and devices provided herein relate to the assembly of highfidelity nucleic acid and nucleic acid libraries having a predefinedsequence using microvolume reactions. More particularly, methods anddevices are provided for polynucleotide synthesis, error filtration,hierarchical assembly, and sequence verification on a solid support. Insome embodiments, picoliter and sub picoliter dispensing anddroplet-moving technologies are applied to access and manipulate theoligonucleotides on DNA microarrays.

BACKGROUND

Using the techniques of recombinant DNA chemistry, it is now common forDNA sequences to be replicated and amplified from nature and thendisassembled into component parts. As component parts, the sequences arethen recombined or reassembled into new DNA sequences. However, relianceon naturally available sequences significantly limits the possibilitiesthat may be explored by researchers. While it is now possible for shortDNA sequences to be directly synthesized from individual nucleosides, ithas been generally impractical to directly construct large segments orassemblies of polynucleotides, i.e., DNA sequences larger than about 400base pairs. Furthermore, chemically synthesized oligonucleotides mayhave an error rate (deletions at a rate of 1 in 100 bases and mismatchesand insertions at about 1 in 400 bases) exceeding the error rateobtainable through enzymatic means of replicating an existing nucleicacid (e.g., PCR). Therefore, there is an urgent need for new technologyto produce high-fidelity polynucleotides.

Oligonucleotide synthesis can be performed through massively parallelcustom syntheses on microchips (Zhou et al. (2004) Nucleic Acids Res.32:5409; Fodor et al. (1991) Science 251:767). However, currentmicrochips have very low surface areas and hence only small amounts ofoligonucleotides can be produced. When released into solution, theoligonucleotides are present at picomolar or lower concentrations persequence, concentrations that are insufficiently high to drivebimolecular priming reactions efficiently. Current methods forassembling small numbers of variant nucleic acids cannot be scaled up ina cost-effective manner to generate large numbers of specified variants.As such, a need remains for improved methods and devices for increasingthroughput and cost-efficiency in high-fidelity gene assembly and thelike.

SUMMARY

Aspects of the technology provided herein relate to devices forpreparing and/or assembling high fidelity polymers. Provided herein aredevices and methods for processing nucleic acid assembly reactions andassembling nucleic acids. It is an object of this invention to providepractical, economical methods of synthesizing custom polynucleotides. Itis a further object to provide a method of producing syntheticpolynucleotides that have lower error rates than syntheticpolynucleotides made by methods known in the art.

In one aspect, the invention provides for methods for assembling apolynucleotide having a predefined sequence from a plurality ofdifferent oligonucleotides, According to one aspect, the methods of theinvention comprise providing a plurality of single-stranded templateoligonucleotides on a support, wherein each of the plurality of templateoligonucleotides comprises a predefined sequence and includes a primerbinding site; generating a complementary oligonucleotide for each of theplurality of template oligonucleotides by enzyme-catalyzed synthesiswithin a first stage or primary droplet, thereby producing a pluralityof double-stranded oligonucleotides; releasing the complementaryoligonuceotides from the double-stranded oligonucleotides into theprimary droplet; combining at least a first and second primary droplets,thereby forming a second stage or secondary droplet, wherein the firstprimary droplet includes a released oligonucleotide that comprises aportion that is complementary to a portion of a released or templateoligonucleotide from the second primary droplet; and exposing thesecondary droplet to conditions suitable for hybridization and ligation,polymerase extension, or polymerase extension and ligation to assemble adouble-stranded polynucleotide having a predefined sequence.

In some embodiments methods of assembling libraries containing nucleicacids having predetermined sequence variations are provided. Assemblystrategies provided herein can be used to generate very large librariesrepresentative of many different nucleic acid sequences of interest.

Methods and devices for analyzing nucleic acid assembly reactions arealso provided herein. In some embodiments, the analysis of the nucleicacid assembly reactions comprises sequencing. In some embodimentsprovided herein, certain microfluidic device configurations may beuseful to amplify, assemble, sequence, isolate and/or purify one or morenucleic acid and/or subassemblies during a nucleic acid assemblyprocedure. In some embodiments of the technology provided herein,hierarchical and/or sequential assembly is performed. In a preferredembodiment, the methods use hierarchical assembly of two or moreoligonucleotides or two or more subassemblies polynucleotide fragmentsat a time. In a further embodiment, the methods use sequential reactionto assemble larger nucleic acids. Oligonucleotides and/or subassemblyfragments may be combined and processed more rapidly and reproducibly toincrease the throughput rate of the assembly.

In some embodiments of the technology provided herein, droplets, asisolated reaction microvolumes, are used for parallel reactions. Methodsand devices provided herein may involve small assembly reaction volumes.For example, reaction volumes of between about 0.5 pL and about 500 nLmay be used. However, smaller or larger volumes may be used. In someembodiments, a mechanical wave actuated dispenser may be used fortransferring volumes of less than 100 nL, less than 10 nL, less than 5nL, less than 100 pL, less than 10 pL, or about 0.5 pL or less. In someembodiments, the mechanical wave actuated dispenser can be apiezoelectric inkjet device or an acoustic liquid handler.

In some embodiments, the throughput rate of an assembly reaction may beincreased by using highly precise droplets dispensing technology.

In some embodiments, a piezoelectric inkjet device or an acoustic liquidhandler may be used to prepare mixtures of reagents or biomolecules(e.g., oligonucleotides) for one or more reactions onto a solidsubstrate or microfluidic substrate. In some embodiments, piezoelectricinkjet or acoustic liquid delivery techniques may be used to introducesamples and/or reagents onto a substrate. In some embodiments, samplesmay be oligonucleotides or polynucleotides. In some embodiments,reagents may be enzymes (e.g. polymerase, ligase, etc.), buffer, dNTPs,primers, etc. or any combination thereof.

In one aspect, methods and devices to amplify oligonucleotides orpolynucleotides in a reaction microvolume or droplet on a solid supportare provided. In some embodiments, oligonucleotides are attached,spotted, immobilized, supported or synthesized on a solid support. Insome embodiments, the oligonucleotides sequences are flanked with primerbinding sites. In preferred embodiments, the oligonucleotides areamplified before being assembled. Oligonucleotides may be amplifiedbefore or after being detached from the solid support and/or eluted in adroplet. In some embodiments, the oligonucleotides are amplified on thesolid support using a scanning laser or a spatial optical modulator(e.g., a digital micromirror device or DMD) capable of individuallymodulating the temperature of a droplet.

In one aspect, methods and devices for isolating nucleic acidintermediates during a polymerase-mediated, a ligation-mediated, orpolymerase and ligation-mediated assembly procedure are provided.Certain assembly steps may generate a mixture of nucleic acids thatinclude a variety of incorrectly assembled nucleic acids.

An assembly reaction mixture may be processed through sequencing and/orselective isolation station arranged to segregate or otherwise groupsubject molecules based on sequence. In preferred embodiments, theassembly reaction mixture or one or more of its components are preparedthrough the action of an acoustic liquid handler or a mechanical waveactuated dispenser (e.g., an acoustic droplet ejector or a piezoelectricinkjet device). In some embodiments, mechanical wave liquid deliverytechnology may be used to directly deposit one or more reagents or areaction mixture directly onto a solid substrate. In some embodiments,the reaction mixture may be generated on the substrate on an appropriatereaction location. In some embodiments, reaction mixtures (e.g.oligonucleotides or subassemblies) in distinct droplets may be mergedtogether by dispensing additional liquid droplets in between or aroundthe original droplets using an acoustic liquid handling technique (e.g.,via the action of a droplet ejector). In some embodiments, the correctlyassembled nucleic acids are removed from the substrate using lasertweezer methods or FACS like methods. Alternatively, incorrectlyassembled or undesired product can be laser ablated. In someembodiments, further assembly steps are performed on the same substrateto assemble larger nucleic acids.

In some embodiments, a sequencing platform that is integrated into thedevices provided herein. In some embodiments, a sequencing station isused to process the products from each different assembly reactions.Certain microfluidic device configurations may be adapted sequencingand/or selective isolation operations within an integrated (e.g.,automated) assembly procedure. Microfluidic devices may be configured toisolate and/or sequence a plurality of assembly reactions rapidly andefficiently. In some embodiments, a plurality of reactions may beprocessed in parallel. Accordingly, the technology provided herein isuseful to increase the rate, yield, and/or precision of nucleic acidassembly. This can decrease the cost and/or delivery time formanufacturing a nucleic acid product.

Accordingly, devices and methods for enhancing the assembly of targetnucleic acids or intermediates thereof are provided herein. In someembodiments, an assembled target nucleic acid may be amplified,sequenced, isolated and/or cloned after it is made. In some embodiments,a host cell may be transformed with the assembled target nucleic acid.The target nucleic acid may be integrated into the genome of the hostcell. In some embodiments, the target nucleic acid may encode apolypeptide. The polypeptide may be expressed (e.g., under the controlof an inducible promoter). The polypeptide may be isolated or purified.A cell transformed with an assembled nucleic acid may be stored,shipped, and/or propagated (e.g., grown in culture).

The invention further provides for methods for synthesizing a pluralityof oligonucleotides having a predefined sequence. According to oneembodiment, the method comprises providing a plurality of support-boundtemplate oligonucleotides in a solution comprising a primer, apolymerase and nucleotides, wherein each of the plurality of templateoligonucleotides comprises a predefined sequence and includes a primerbinding site, and wherein the primer comprises at least one nucleaserecognition site; exposing the plurality of template oligonucleotides toconditions suitable for primer hybridization and polymerase extension,thereby extending the primers to produce a complementary oligonucleotidefor each of the plurality of template oligonucleotides; releasing thecomplementary oligonuceotides; exposing the complementaryoligonucleotides to a nuclease under conditions suitable for thenuclease to bind to the nuclease recognition site on the primer andcleave the primer from complementary oligonucleotide; and exposing thecomplementary and template oligonucleotides to conditions suitable forhybridization; thereby to produce a plurality of partiallydouble-stranded oligonucleotides. In one embodiment, methods of theinvention further comprise washing the plurality of partiallydouble-stranded oligonucleotides and releasing the complementaryoligonucleotides. Aspects of the invention also contemplate using aprimer comprising at least two nuclease recognition sites, and the stepof exposing the cleaved primer to a second nuclease under conditionssuitable for the second nuclease to bind to the primer and subject theprimer to further cleavage.

Other features and advantages of the devices and methods provided hereinwill be apparent from the following detailed description, and from theclaims. The claims provided below are hereby incorporated into thissection by reference. The various embodiments described herein can becomplimentary and can be combined or used together in a mannerunderstood by the skilled person in view of the teachings containedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates an embodiment of the device comprising a source wellplate (101), containing the reagents for reactions, a solid support(102), with solid-attached surface or supported molecules, a transducer(103), a coupling fluid (104), reagents (105) inside a well, solidattached or surface supported molecules (110), surface droplets (111)formed by the dispensed droplets (106), a “merge” droplet (112)dispensed between two surface droplets (111), a surface droplet (170)dispensed for the purpose of alignment of the droplet to the solidattached molecules (111), an electronics camera (171) and used toprovide physical registration (positioning) and a surface mark (172)fixed on the solid support (102).

FIG. 1B illustrates an embodiment using an inkjet device for dropletdispensing. The head assembly (180) includes multiple jetting modules(181), with each module containing at least one reservoir (183) havingat least one inlet (184). Each jetting module can have one or more thanone nozzle (182), which can be arranged in an 1D or 2D array to form anozzle pattern. The nozzles can have well defined dimensions to allowdroplets (106) to form under the influence of a mechanical wavegenerated by a transducer (185). Each nozzle in a jetting module can becontrolled by an independent transducer. Alternatively, multiple nozzlescan be controlled by a common transducer. The head assembly can haveone, or two, or three degrees of freedom to move in physical space. Thetransducers can be controlled, for example, by electronics that are incommunication with a computer.

FIG. 2 illustrates an embodiment of a solid support comprising differentand unique molecules (201, 202, 203, 204) supported or attached to thesurface of 102, a unique molecule (250) supported or attached to thesurface of 102 at multiple positions other unique molecules (299)supported or attached to the surface of 102.

FIG. 3A illustrates an embodiment of a solid support comprisingdifferent molecules (A, B, C, etc.) and a non-limiting example of anassembly strategy. FIG. 3B illustrates a non-limiting example of anassembly strategy. FIG. 3C illustrates a non limiting example of ahierarchical assembly strategy. FIG. 3D illustrates a non limitingexample of a hierarchical assembly strategy.

FIG. 4 illustrates an embodiment of thermal control device and procedurecomprising solid support substrate (401) comprising immobilizedmolecules (404); an optical absorbent material (402) in the surfacedroplet (403), the surface droplet comprising molecules (409) insolution, an optical absorbent material (405) on the surface of 401, anoptical energy source (406), a scanning setup (407), energy beams (408)and a plurality of reaction sites (420, 421, 422, 423). An opticallyabsorbing material (405) (e.g., a dye) can be added to the surfacedroplet (403) (e.g., reaction volume).

FIG. 5A illustrates non-limiting example of method and devices tocapture desired product comprising a microfluidics reaction chamber(701) for sequencing reaction; individual sequencing reaction sites,containing undesirable material (702), reaction site containing desiredpopulation (703), an outlet (exit) (799) of the chamber, a Laser. FIG.5B illustrates a non-limiting example of device using an optical tweezersystem and comprising a lens system (750) to implement optical tweezersand a location of the focus of an optical tweezers system (751). FIG. 5Cillustrates a non-limiting example using a laser device to ablateundesired products comprising a high power laser (761) to generateoptical energy, a scanning setup (762) to control the position of theenergy beam at specific location (763). FIG. 5D illustrates anon-limiting example to track desired product suing a vision systemcomprising: Outlet of the reaction chamber (701); 772: An input flow toinduce the contents of 701 to exit at 771; 773: An offshoot channel thatcontains the undesirable material; 774: An offshoot channel thatcontains the desirable material; 775: A detector that determines fixateson the desired products, and tracking its position; 776: Flowcontrollers to direct the sorting process; and 777: Sorting junction.FIG. 5E illustrates a method to achieve “selective isolation” that caninvolve the utilization of photopolymers to retain and trap desirableproducts.

FIG. 6 illustrates an exemplary structure of a dentron molecule.

FIG. 7A illustrates a DNA library on a microarray. FIG. 7B illustratesthat picoliter and sub picoliter volume droplets can be used to accessthe large library of material on a DNA microarray.

FIG. 8 illustrates an exemplary method of error filtration.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are apparatuses for preparing and/or assemblingpolymers. In some embodiments, devices and methods for processingnucleic acid assembly reactions and assembling nucleic acids areprovided herein. As used herein the term “nucleic acid” “polynucleotide”and “oligonucleotide” are used interchangeably and refers to polymericform of nucleotides, either ribonucleotides and/or deoxyribonucleotidesor a modified form of either type of nucleotides. The term should beunderstood to include equivalents, analogs of either RNA or DNA madefrom nucleotide analogs and as applicable to the embodiment beingdescribed, single stranded or double stranded polynucleotides.

In some embodiments, methods of assembling libraries containing nucleicacids having predetermined sequence variations are provided herein.Assembly strategies provided herein can be used to generate very largelibraries representative of many different nucleic acid sequences ofinterest. In some embodiments, libraries of nucleic acid are librariesof sequence variants. Sequence variants may be variants of a singlenaturally-occurring protein encoding sequence. However, in someembodiments, sequence variants may be variants of a plurality ofdifferent protein-encoding sequences.

Accordingly, one aspect of the technology provided herein relates to thedesign of assembly strategies for preparing precise high-density nucleicacid libraries. Another aspect of the technology provided herein relatesto assembling precise high-density nucleic acid libraries. Aspects ofthe technology provided herein also provide precise high-density nucleicacid libraries. A high-density nucleic acid library may include morethat 100 different sequence variants (e.g., about 10² to 10³; about 10³to 10⁴; about 10⁴ to 10⁵; about 10⁵ to 10⁶; about 10⁶ to 10⁷; about 10⁷to 10⁸; about 10⁸ to 10⁹; about 10⁹ to 10¹⁰; about 10¹⁰ to 10¹¹; about10¹¹ to 10¹²; about 10¹² to 10¹³; about 10¹³ to 10¹⁴; about 10¹⁴ to10¹⁵; or more different sequences) wherein a percentage of the differentsequences are specified sequences as opposed to random sequences (e.g.,more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the sequences arepredetermined sequences of interest).

Provided herein are microfluidic devices and systems for preparingand/or assembling polymers. One aspect of the technology provided hereinis generally directed to the synthesis of long polymers and biopolymerssuch as nucleic acids. Aspects of the technology provided herein may beuseful for increasing the accuracy, yield, throughput, and/or costefficiency of nucleic acid assembly reactions.

In some embodiments, the assembly procedure may include several paralleland/or sequential reaction steps in which a plurality of differentnucleic acids or oligonucleotides are synthesized or immobilized,amplified, filtered, and combined for assembly (e.g., by extension orligation as described herein) to generate a longer nucleic acid productto be used for further assembly, cloning, or other applications.Assembly strategies provided herein can be used to generate very largelibraries representative of many different nucleic acid sequences ofinterest.

In some embodiments of the technology provided herein, immobilized orsurface-supported oligonucleotides or polynucleotides are used as asource of material. In various embodiments, the methods described hereinuses oligonucleotides, their sequence being determined based on thesequence of the final polynucleotides constructs to be synthesized. Inone embodiment, “oligonucleotides” are short nucleic acid molecules. Forexample, oligonucleotides may be from 10 to about 300 nucleotides, from20 to about 400 nucleotides, from 30 to about 500 nucleotides, from 40to about 600 nucleotides, or more than about 600 nucleotides long.However shorter or longer oligonucleotides may be used. Oligonucleotidesmay be designed to have different length. In some embodiments, thesequence of the polynucleotide construct may be divided up into aplurality of shorter sequences that can be synthesized in parallel andassembled into a single or a plurality of desired polynucleotideconstructs using the methods described herein. In certain embodiments,the oligonucleotides are designed to provide the full sense andantisense strands of the polynucleotide construct. After hybridizationof the plus and minus strand oligonucleotides, two double strandedoligonucleotides are subjected to ligation or polymerization in order toform a first subassembly product. Subassembly products are thensubjected to ligation or polymerization to form a larger DNA or the fullDNA sequence.

Ligase-based assembly techniques may involve one or more suitable ligaseenzymes that can catalyze the covalent linking of adjacent 3′ and 5′nucleic acid termini (e.g., a 5′ phosphate and a 3′ hydroxyl of nucleicacid(s) annealed on a complementary template nucleic acid such that the3′ terminus is immediately adjacent to the 5′ terminus). Accordingly, aligase may catalyze a ligation reaction between the 5′ phosphate of afirst nucleic acid to the 3′ hydroxyl of a second nucleic acid if thefirst and second nucleic acids are annealed next to each other on atemplate nucleic acid). A ligase may be obtained from recombinant ornatural sources. A ligase may be a heat-stable ligase. In someembodiments, a thermostable ligase from a thermophilic organism may beused. Examples of thermostable DNA ligases include, but are not limitedto: Tth DNA ligase (from Thermus thermophilus, available from, forexample, Eurogentec and GeneCraft); Pfu DNA ligase (a hyperthermophilicligase from Pyrococcus furiosus); Taq ligase (from Thermus aquaticus),any other suitable heat-stable ligase, or any combination thereof. Insome embodiments, one or more lower temperature ligases may be used(e.g., T4 DNA ligase). A lower temperature ligase may be useful forshorter overhangs (e.g., about 3, about 4, about 5, or about 6 baseoverhangs) that may not be stable at higher temperatures.

Non-enzymatic techniques can be used to ligate nucleic acids. Forexample, a 5′-end (e.g., the 5′ phosphate group) and a 3′-end (e.g., the3′ hydroxyl) of one or more nucleic acids may be covalently linkedtogether without using enzymes (e.g., without using a ligase). In someembodiments, non-enzymatic techniques may offer certain advantages overenzyme-based ligations. For example, non-enzymatic techniques may have ahigh tolerance of non-natural nucleotide analogues in nucleic acidsubstrates, may be used to ligate short nucleic acid substrates, may beused to ligate RNA substrates, and/or may be cheaper and/or more suitedto certain automated (e.g., high throughput) applications.

Non-enzymatic ligation may involve a chemical ligation. In someembodiments, nucleic acid termini of two or more different nucleic acidsmay be chemically ligated. In some embodiments, nucleic acid termini ofa single nucleic acid may be chemically ligated (e.g., to circularizethe nucleic acid). It should be appreciated that both strands at a firstdouble-stranded nucleic acid terminus may be chemically ligated to bothstrands at a second double-stranded nucleic acid terminus. However, insome embodiments only one strand of a first nucleic acid terminus may bechemically ligated to a single strand of a second nucleic acid terminus.For example, the 5′ end of one strand of a first nucleic acid terminusmay be ligated to the 3′ end of one strand of a second nucleic acidterminus without the ends of the complementary strands being chemicallyligated.

Accordingly, a chemical ligation may be used to form a covalent linkagebetween a 5′ terminus of a first nucleic acid end and a 3′ terminus of asecond nucleic acid end, wherein the first and second nucleic acid endsmay be ends of a single nucleic acid or ends of separate nucleic acids.In one aspect, chemical ligation may involve at least one nucleic acidsubstrate having a modified end (e.g., a modified 5′ and/or 3′ terminus)including one or more chemically reactive moieties that facilitate orpromote linkage formation. In some embodiments, chemical ligation occurswhen one or more nucleic acid termini are brought together in closeproximity (e.g., when the termini are brought together due to annealingbetween complementary nucleic acid sequences). Accordingly, annealingbetween complementary 3′ or 5′ overhangs (e.g., overhangs generated byrestriction enzyme cleavage of a double-stranded nucleic acid) orbetween any combination of complementary nucleic acids that results in a3′ terminus being brought into close proximity with a 5′ terminus (e.g.,the 3′ and 5′ termini are adjacent to each other when the nucleic acidsare annealed to a complementary template nucleic acid) may promote atemplate-directed chemical ligation. Examples of chemical reactions mayinclude, but are not limited to, condensation, reduction, and/orphoto-chemical ligation reactions. It should be appreciated that in someembodiments chemical ligation can be used to produce naturally occurringphosphodiester internucleotide linkages, non-naturally-occurringphosphamide pyrophosphate internucleotide linkages, and/or othernon-naturally-occurring internucleotide linkages.

In some embodiments, the process of chemical ligation may involve one ormore coupling agents to catalyze the ligation reaction. A coupling agentmay promote a ligation reaction between reactive groups in adjacentnucleic acids (e.g., between a 5′-reactive moiety and a 3′-reactivemoiety at adjacent sites along a complementary template). In someembodiments, a coupling agent may be a reducing reagent (e.g.,ferricyanide), a condensing reagent such (e.g., cyanoimidazole, cyanogenbromide, carbodiimide, etc.), or irradiation (e.g., UV irradiation forphoto-ligation).

In some embodiments, a chemical ligation may be an autoligation reactionthat does not involve a separate coupling agent. In autoligation, thepresence of a reactive group on one or more nucleic acids may besufficient to catalyze a chemical ligation between nucleic acid terminiwithout the addition of a coupling agent (see, for example, Xu et al.,(1997) Tetrahedron Lett. 38:5595-8). Non-limiting examples of thesereagent-free ligation reactions may involve nucleophilic displacementsof sulfur on bromoacetyl, tosyl, or iodo-nucleoside groups (see, forexample, Xu et al., (2001) Nat. Biotech. 19:148-52). Nucleic acidscontaining reactive groups suitable for autoligation can be prepareddirectly on automated synthesizers (see, for example, Xu et al., (1999)Nuc. Acids Res. 27:875-81). In some embodiments, a phosphorothioate at a3′ terminus may react with a leaving group (such as tosylate or iodide)on a thymidine at an adjacent 5′ terminus. In some embodiments, twonucleic acid strands bound at adjacent sites on a complementary targetstrand may undergo auto-ligation by displacement of a 5′-end iodidemoiety (or tosylate) with a 3′-end sulfur moiety. Accordingly, in someembodiments the product of an autoligation may include anon-naturally-occurring internucleotide linkage (e.g., a single oxygenatom may be replaced with a sulfur atom in the ligated product).

In some embodiments, a synthetic nucleic acid duplex can be assembledvia chemical ligation in a one step reaction involving simultaneouschemical ligation of nucleic acids on both strands of the duplex. Forexample, a mixture of 5′-phosphorylated oligonucleotides correspondingto both strands of a target nucleic acid may be chemically ligated by a)exposure to heat (e.g., to 97° C.) and slow cooling to form a complex ofannealed oligonucleotides, and b) exposure to cyanogen bromide or anyother suitable coupling agent under conditions sufficient to chemicallyligate adjacent 3′ and 5′ ends in the nucleic acid complex.

In some embodiments, a synthetic nucleic acid duplex can be assembledvia chemical ligation in a two step reaction involving separate chemicalligations for the complementary strands of the duplex. For example, eachstrand of a target nucleic acid may be ligated in a separate reactioncontaining phosphorylated oligonucleotides corresponding to the strandthat is to be ligated and non-phosphorylated oligonucleotidescorresponding to the complementary strand. The non-phosphorylatedoligonucleotides may serve as a template for the phosphorylatedoligonucleotides during a chemical ligation (e.g., using cyanogenbromide). The resulting single-stranded ligated nucleic acid may bepurified and annealed to a complementary ligated single-stranded nucleicacid to form the target duplex nucleic acid (see, for example, Shabarovaet al., (1991) Nucl. Acids Res. 19:4247-51).

In one aspect, a nucleic acid fragment may be assembled in a polymerasemediated assembly reaction from a plurality of oligonucleotides that arecombined and extended in one or more rounds of polymerase-mediatedextensions. In some embodiments, the oligonucleotides are overlappingoligonucleotides covering the full sequence but leaving single strandedgaps that may be filed in by chain extension. The plurality of differentoligonucleotides may provide either positive sequences, negativesequences, or a combination of both positive and negative sequencescorresponding to the entire sequence of the nucleic acid fragment to beassembled. In some embodiments, one or more different oligonucleotidesmay have overlapping sequence regions (e.g., overlapping 5′ regions oroverlapping 3′ regions). Overlapping sequence regions may be identical(i.e., corresponding to the same strand of the nucleic acid fragment) orcomplementary (i.e., corresponding to complementary strands of thenucleic acid fragment). The plurality of oligonucleotides may includeone or more oligonucleotide pairs with overlapping identical sequenceregions, one or more oligonucleotide pairs with overlappingcomplementary sequence regions, or a combination thereof. Overlappingsequences may be of any suitable length. For example, overlappingsequences may encompass the entire length of one or more nucleic acidsused in an assembly reaction. Overlapping sequences may be between about5 and about 500 oligonucleotides long (e.g., between about 10 and 100,between about 10 and 75, between about 10 and 50, about 20, about 25,about 30, about 35, about 45, about 50, etc.). However, shorter, longer,or intermediate overlapping lengths may be used. It should beappreciated that overlaps between different input nucleic acids used inan assembly reaction may have different lengths.

Polymerase-based assembly techniques may involve one or more suitablepolymerase enzymes that can catalyze a template-based extension of anucleic acid in a 5′ to 3′ direction in the presence of suitablenucleotides and an annealed template. A polymerase may be thermostable.A polymerase may be obtained from recombinant or natural sources. Insome embodiments, a thermostable polymerase from a thermophilic organismmay be used. In some embodiments, a polymerase may include a 3′→5′exonuclease/proofreading activity. In some embodiments, a polymerase mayhave no, or little, proofreading activity (e.g., a polymerase may be arecombinant variant of a natural polymerase that has been modified toreduce its proofreading activity). Examples of thermostable DNApolymerases include, but are not limited to: Taq (a heat-stable DNApolymerase from the bacterium Thermus aquaticus); Pfu (a thermophilicDNA polymerase with a 3′→5′ exonuclease/proofreading activity fromPyrococcus furiosus, available from for example Promega); VentR® DNAPolymerase and VentRO (exo-) DNA Polymerase (thermophilic DNApolymerases with or without a 3′→5′ exonuclease/proofreading activityfrom Thermococcus litoralis; also known as Th polymerase); Deep VentR®DNA Polymerase and Deep VentR® (exo-) DNA Polymerase (thermophilic DNApolymerases with or without a 3′→5′ exonuclease/proofreading activityfrom Pyrococcus species GB-D; available from New England Biolabs); KODHiFi (a recombinant Thermococcus kodakaraensis KODI DNA polymerase witha 3′→5′ exonuclease/proofreading activity, available from Novagen,);BIO-X-ACT (a mix of polymerases that possesses 5′-3′ DNA polymeraseactivity and 3′→5′ proofreading activity); Klenow Fragment (anN-terminal truncation of E. coli DNA Polymerase I which retainspolymerase activity, but has lost the 5′→3′ exonuclease activity,available from, for example, Promega and NEB); Sequenase™ (T7 DNApolymerase deficient in T-5′ exonuclease activity); Phi29 (bacteriophage29 DNA polymerase, may be used for rolling circle amplification, forexample, in a TempliPhi™ DNA Sequencing Template Amplification Kit,available from Amersham Biosciences); TopoTaq (a hybrid polymerase thatcombines hyperstable DNA binding domains and the DNA unlinking activityof Methanopyrus topoisomerase, with no exonuclease activity, availablefrom Fidelity Systems); TopoTaq HiFi which incorporates a proofreadingdomain with exonuclease activity; Phusion™ (a Pyrococcus-like enzymewith a processivity-enhancing domain, available from New EnglandBiolabs); any other suitable DNA polymerase, or any combination of twoor more thereof.

In some embodiments, the polymerase can be a SDP (strand-displacingpolymerase; e.g, an SDPe—which is an SDP with no exonuclease activity).This allows isothermal PCR (isothermal extension, isothermalamplification) where duplication of a template takes place at a uniformtemperature. As the polymerase (for example, Phi29, Bst) travels along atemplate it displaces the complementary strand (e.g., created inprevious extension reactions). As the displaced DNAs are singlestranded, primers can bind at a consistent temperature, removing theneed for any thermocycling during amplification, thereby avoiding ordecreasing evaporation of the reaction mixture.

It should be appreciated that the description of the assembly reactionsin the context of the oligonucleotides is not intended to be limiting.For example, other polynucleotides (e.g. single stranded,double-stranded polynucleotides, restriction fragments, amplificationproducts, naturally occurring polynucleotides, etc.) may be included inan assembly reaction, along with one or more oligonucleotides, in orderto generate a polynucleotide of interest.

In some embodiments, the oligonucleotides may comprise universal (commonto all oligonucleotides), semiuniversal (common to at least of portionof the oligonucleotides) or individual or unique primer (specific toeach oligonucleotide) binding sites on either the 5′ end or the 3′ endor both. As used herein, the term “universal” primer or primer bindingsite means that a sequence used to amplify the oligonucleotide is commonto all oligonucleotides such that all such oligonucleotides can beamplified using a single set of universal primers. In othercircumstances, an oligonucleotide contains a unique primer binding site.As used herein, the term “unique primer binding site” refers to a set ofprimer recognition sequences that selectively amplifies a subset ofoligonucleotides. In yet other circumstances, an oligonucleotidecontains both universal and unique amplification sequences, which canoptionally be used sequentially.

In some embodiments, primers/primer binding site may be designed to betemporary. For example, temporary primers may be removed by chemical,light based or enzymatic cleavage. For example, primers/primer bindingsites may be designed to include a restriction endonuclease cleavagesite. In an exemplary embodiment, a primer/primer binding site containsa binding and/or cleavage site for a type IIs restriction endonuclease.In such case, amplification sequences may be designed so that once adesired set of oligonucleotides is amplified to a sufficient amount, itcan then be cleaved by the use of an appropriate type IIs restrictionenzyme that recognizes an internal type IIs restriction enzyme sequenceof the oligonucleotide.

In some embodiments, after amplification, the pool of nucleic acids maybe contacted with one or more endonucleases to produce double strandedbreaks thereby removing the primers/primer binding sites. In certainembodiments, the forward and reverse primers may be removed by the sameor different restriction endonucleases. Any type of restrictionendonuclease may be used to remove the primers/primer binding sites fromnucleic acid sequences. A wide variety of restriction endonucleaseshaving specific binding and/or cleavage sites are commerciallyavailable, for example, from New England Biolabs (Beverly, Mass.). Invarious embodiments, restriction endonucleases that produce 3′overhangs, 5′ overhangs or blunt ends may be used. When using arestriction endonuclease that produces an overhang, an exonuclease(e.g., RecJ_(f), Exonuclease I, Exonuclease T, S₁ nuclease, P₁ nuclease,mung bean nuclease, T4 DNA polymerase, CEL I nuclease, etc.) may be usedto produce blunt ends. Alternatively, the sticky ends formed by thespecific restriction endonuclease may be used to facilitate assembly ofsubassemblies in a desired arrangement. In an exemplary embodiment, aprimer/primer binding site that contains a binding and/or cleavage sitefor a type IIs restriction endonuclease may be used to remove thetemporary primer.

The term “type-IIs restriction endonuclease” refers to a restrictionendonuclease having a non-palindromic recognition sequence and acleavage site that occurs outside of the recognition site (e.g., from 0to about 20 nucleotides distal to the recognition site). Type IIsrestriction endonucleases may create a nick in a double stranded nucleicacid molecule or may create a double stranded break that produces eitherblunt or sticky ends (e.g., either 5′ or 3′ overhangs). Examples of TypeIIs endonucleases include, for example, enzymes that produce a 3′overhang, such as, for example, Bsr I, Bsm I, BstF5 I, BsrD I, Bts I,Mnl I, BciV I, Hph I, Mbo II, Eci I, Acu I, Bpm I, Mme I, BsaX I, Bcg I,Bae I, Bfi I, TspDT I, TspGW I, Taq II, Eco57 I, Eco57M I, Gsu I, Ppi I,and Psr I; enzymes that produce a 5′ overhang such as, for example, BsmAI, Ple I, Fau I, Sap I, BspM I, SfaN I, Hga I, Bvb I, Fok I, BceA I,BsmF I, Ksp632 I, Eco31 I, Esp3 I, Aar I; and enzymes that produce ablunt end, such as, for example, Mly I and Btr I. Type-IIs endonucleasesare commercially available and are well known in the art (New EnglandBiolabs, Beverly, Mass.).

Some embodiments of the devices and methods provided herein useoligonucleotides that are immobilized on a solid support. A solidsupport refers to a porous or non-porous solvent insoluble material. Asused herein “porous” means that the material contains pores havingsubstantially uniform diameters (for example in the nm range). Porousmaterials include paper, synthetic filters etc. In such porousmaterials, the reaction may take place within the pores. The support canhave any one of a number of shapes, such as pin, strip, plate, disk,rod, cylindrical structure, particle, including bead, and the like. Thesupport can be hydrophilic or capable of being rendered hydrophilic andincludes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly (4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), polyvinylidene difluoride (PVDF) membrane, glass,controlled pore glass, magnetic controlled pore glass, ceramics, metals,and the like etc.; either used by themselves or in conjunction withother materials.

In some embodiments, oligonucleotides are synthesized on an arrayformat. For example, single stranded oligonucleotides are synthesized insitu on a common support wherein each oligonucleotide is synthesized ona separate feature (or spot) on the substrate. It should be appreciatedthat each oligonucleotide fragment can be of any length, but istypically 10-400 bases long. Arrays may be constructed, custom orderedor purchased from a commercial vendor (e.g., Agilent, Affymetrix,Nimblegen). Various methods of construction are well known in the arte.g. maskless array synthesizers, light directed methods utilizingmasks, flow channel methods, spotting methods etc.

In some embodiments, construction and/or selection oligonucleotides maybe synthesized on a solid support using maskless array synthesizer(MAS). Maskless array synthesizers are described, for example, in PCTapplication No. WO 99/42813 and in corresponding U.S. Pat. No.6,375,903. Other examples are known of maskless instruments which canfabricate a custom DNA microarray in which each of the features in thearray has a single stranded DNA molecule of desired sequence.

Other methods for synthesizing construction and/or selectionoligonucleotides include, for example, light-directed methods utilizingmasks, flow channel methods, spotting methods, pin-based methods, andmethods utilizing multiple supports.

Light directed methods utilizing masks (e.g., VLSIPS™ methods) for thesynthesis of oligonucleotides is described, for example, in U.S. Pat.Nos. 5,143,854, 5,510,270 and 5,527,681. These methods involveactivating predefined regions of a solid support and then contacting thesupport with a preselected monomer solution. Selected regions can beactivated by irradiation with a light source through a mask much in themanner of photolithography techniques used in integrated circuitfabrication. Other regions of the support remain inactive becauseillumination is blocked by the mask and they remain chemicallyprotected. Thus, a light pattern defines which regions of the supportreact with a given monomer. By repeatedly activating different sets ofpredefined regions and contacting different monomer solutions with thesupport, a diverse array of polymers is produced on the support. Othersteps, such as washing unreacted monomer solution from the support, canbe optionally used. Other applicable methods include mechanicaltechniques such as those described in U.S. Pat. No. 5,384,261.

Additional methods applicable to synthesis of construction and/orselection oligonucleotides on a single support are described, forexample, in U.S. Pat. No. 5,384,261. For example, reagents may bedelivered to the support by either (1) flowing within a channel definedon predefined regions or (2) “spotting” on predefined regions. Otherapproaches, as well as combinations of spotting and flowing, may beemployed as well. In each instance, certain activated regions of thesupport are mechanically separated from other regions when the monomersolutions are delivered to the various reaction sites. Flow channelmethods involve, for example, microfluidic systems to control synthesisof oligonucleotides on a solid support. For example, diverse polymersequences may be synthesized at selected regions of a solid support byforming flow channels on a surface of the support through whichappropriate reagents flow or in which appropriate reagents are placed.Spotting methods for preparation of oligonucleotides on a solid supportinvolve delivering reactants in relatively small quantities by directlydepositing them in selected regions. In some steps, the entire supportsurface can be sprayed or otherwise coated with a solution, if it ismore efficient to do so. Precisely measured aliquots of monomersolutions may be deposited dropwise by a dispenser that moves fromregion to region.

Pin-based methods for synthesis of oligonucleotides on a solid supportare described, for example, in U.S. Pat. No. 5,288,514. Pin-basedmethods utilize a support having a plurality of pins or otherextensions. The pins are each inserted simultaneously into individualreagent containers in a tray. An array of 96 pins is commonly utilizedwith a 96-container tray, such as a 96-well microtitre dish. Each trayis filled with a particular reagent for coupling in a particularchemical reaction on an individual pin. Accordingly, the trays willoften contain different reagents. Since the chemical reactions have beenoptimized such that each of the reactions can be performed under arelatively similar set of reaction conditions, it becomes possible toconduct multiple chemical coupling steps simultaneously.

In yet another embodiment, a plurality of construction and/or selectionoligonucleotides may be synthesized on multiple supports. On example isa bead based synthesis method which is described, for example, in U.S.Pat. Nos. 5,770,358; 5,639,603; and 5,541,061. For the synthesis ofmolecules such as oligonucleotides on beads, a large plurality of beadsis suspended in a suitable carrier (such as water) in a container. Thebeads are provided with optional spacer molecules having an active siteto which is complexed, optionally, a protecting group. At each step ofthe synthesis, the beads are divided for coupling into a plurality ofcontainers. After the nascent oligonucleotide chains are deprotected, adifferent monomer solution is added to each container, so that on allbeads in a given container, the same nucleotide addition reactionoccurs. The beads are then washed of excess reagents, pooled in a singlecontainer, mixed and re-distributed into another plurality of containersin preparation for the next round of synthesis. It should be noted thatby virtue of the large number of beads utilized at the outset, therewill similarly be a large number of beads randomly dispersed in thecontainer, each having a unique oligonucleotide sequence synthesized ona surface thereof after numerous rounds of randomized addition of bases.An individual bead may be tagged with a sequence which is unique to thedouble-stranded oligonucleotide thereon, to allow for identificationduring use.

Pre-synthesized oligonucleotide and/or polynucleotide sequences may beattached to a support or synthesized in situ using light-directedmethods, flow channel and spotting methods, inkjet methods, pin-basedmethods and bead-based methods set forth in the following references:McGall et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:13555; SyntheticDNA Arrays In Genetic Engineering, Vol. 20:111, Plenum Press (1998);Duggan et al. (1999) Nat. Genet. S21:10; Microarrays: Making Them andUsing Them In Microarray Bioinformatics, Cambridge University Press,2003; U.S. Patent Application Publication Nos. 2003/0068633 and2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890, 6,824,866, 6,800,439,6,375,903 and 5,700,637; and PCT Publication Nos. WO 04/031399, WO04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO 03/065038, WO03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO 03/040410 and WO02/24597; incorporated herein by reference in their entirety for allpurposes. In a preferred embodiment, pre-synthesized are attached to asupport or are synthesized using a spotting methodology wherein monomerssolutions are deposited dropwise by a dispenser that move from region toregion (e.g. ink jet). In some embodiments, oligonucleotides are spottedon a solid support using, for example, a mechanical wave actuateddispenser.

In some embodiments of the technology provided herein, polynucleotidesare assembled by multiplex nucleic acid assembly. Multiplex nucleic acidassembly relates to the assembly of a plurality of oligonucleotidesand/or polynucleotides to generate a longer nucleic acid product. Thepolynucleotide product may be at least about 1, 2, 3, 4, 5, 8, 10, 15,20, 25, 30, 40, 50, 75, or 100 kilobases (kb), or 1 megabase (mb), orlonger. In some embodiments, oligonucleotides or polynucleotides areassembled via a hierarchical and sequential approach. Oligonucleotidesor polynucleotides to be assembled are synthesized or spotted onadjacent features or spots on the support. For example, twooligonucleotides having complementary regions may be immobilized orsynthesized in close proximity to each other. Spot or features orlocation may be placed every 5 μm, 10 μm, 25 μm, 50 μm, 75 μm or above.However smaller or larger spacing may be used. In some embodiments, thesolid support has 1000 to 10⁶ features or active sites.

It should be appreciated that the description of the assembly reactionsof oligonucleotides that are supported on adjacent features on thesupport is not intended to be limiting. For example, additionaldifferent synthetic oligonucleotides or polynucleotides (e.g. singlestranded or double stranded oligonucleotides) may be included in anassembly reaction together with solid-supported oligonucleotides inorder to generate a polynucleotide of interest. The additional syntheticoligonucleotide can be prepared by conventional phosphoramiditechemistry. Phosphoramidite chemistry can be carried out usingcommercially available machines as is available, for example, fromIntegrated DNA Technologies.

In some embodiments, reagents are spotted or jetted onto the solidsupport. Droplets containing enzymes, dNTP, buffer, primers or anycombination thereof are spotted to match the DNA spots on themicroarray. In other embodiments, the solution deposited on the solidsupport comprises one or more different and unique oligonucleotidemolecules.

In various embodiments, a mechanical wave actuated dispenser can be usedfor transferring small volumes of fluids (e.g., nanoliter, picoliter, orsub-picoliter). A mechanical wave actuated dispenser can be apiezoelectric inkjet device or an acoustic liquid handler. Apiezoelectric inkjet device can eject fluids by actuating apiezoelectric actuation mechanism, which forces fluid droplets to beejected. Piezoelectrics in general have good operating bandwidth and cangenerate large forces in a compact size. Some of the commerciallyavailable piezoelectric inkjet microarraying instruments include thosefrom Perkin Elmer (Wellesley, Mass.), GeSim (Germany) and MicroFab(Plano, Tex.). Typical piezoelectric dispensers can create droplets inthe picoliter range and with coefficient of variations of 3-7%.Inkjetting technologies and devices for ejecting a plurality of fluiddroplets toward discrete sites on a substrate surface for depositionthereon have been described in a number of patents such as U.S. Pat.Nos. 6,511,849; 6,514,704; 6,042,211; 5,658,802, the disclosure of whichare incorporated herein by reference.

In one embodiment, the fluid or solution deposition is performed usingan acoustic liquid handler or ejector. Acoustic devices are non-contactdispensing devices able to dispense small volume of fluid (e.g.picoliter to microliter), see for example Echo 550 from Labcyte (CA),HTS-01 from EDC Biosystems. Acoustic technologies and devices foracoustically ejecting a plurality of fluid droplets toward discretesites on a substrate surface for deposition thereon have been describedin a number of patents such as U.S. Pat. Nos. 6,416,164; 6,596,239;6,802,593; 6,932,097; 7,090,333 and US Patent Application 2002-0037579,the disclosure of which are incorporated herein by reference. Theacoustic device includes an acoustic radiation generator or transducerthat may be used to eject fluid droplets from a reservoir (e.g.microplate wells) through a coupling medium. The pressure of the focusedacoustic waves at the fluid surface creates an upwelling, therebycausing the liquid to urge upwards so as to eject a droplet, for examplefrom a well of a source plate, to a receiving plate positioned above thefluid reservoir. The volume of the droplet ejected can be determined byselecting the appropriate sound wave frequency. Methods and devicesprovided herein preferably involve small assembly reaction volumes ormicrovolumes. One should appreciate that the shape of small volumes ofliquids is governed and maintained by the surface tension of the liquid.In some embodiments, the microvolume is bounded completely or almostcompletely by free surface forming a droplet or microdrop. In apreferred embodiment, the assembly reaction microvolume may be in theform of a droplet. In some embodiments, reaction microvolumes of betweenabout 0.5 pL and about 100 nL may be used. However, smaller or largervolumes may be used. In some embodiments, a mechanical wave actuateddispenser may be used for transferring volumes of less than 100 nL, lessthan 10 nL, less than 5 nL, less than 100 pL, less than 10 pL, or about0.5 pL or less. In some embodiments, the mechanical wave actuateddispenser can be a piezoelectric inkjet device or an acoustic liquidhandler.

In some embodiments, the source plate comprising primers, master mix,release chemicals, enzymes, or any combination thereof and thedestination plates comprising the oligonucleotides or polynucleotidesare matched up to allow proper delivery or spotting of the reagent tothe proper site. The mechanical wave actuated dispenser may be coupledwith a microscope and/or a camera to provide positional selection ofdeposited spots. A camera may be placed on both sides of the destinationplate or substrate. A camera may be used to register to the positioningon the array especially if the DNA is coupled with a fluorescent label.As shown in FIG. 1A and described below components of the deviceinclude: 101: Source well plate; containing the reagents for reactions,this element can travel in at least 2 degree-of-freedom (>2DOF); 102:Solid support, with solid attached molecules, this element can travel inat least 2 degree-of-freedom (>2DOF); 103: Transducer, to create amechanical wave which causes droplets to form and travel, this elementcan travel in at least 2 degree-of-freedom (>2DOF); 104: Coupling fluid,to allow the mechanical wave to couple to the well plate (101); 105:Reagents inside a well on the source well plate (101); 171: A camera(such as an electronic camera) used to provide physical registration(positioning) and 172: A surface mark fixed on the solid support (102)to provide a reference position on the solid support. FIG. 1B showsanother example where an inkjet device 180 (e.g., piezoelectric) can beused to dispense droplets.

A mechanical wave actuated dispenser (103, 180) can be used to createtraveling droplets (106) from a reagent source (101). The createdtraveling droplets (106) can be deposited onto a receiving surface, inthis case a solid support (102). The position of the deposited droplets(111) on the solid support (102) can be controlled by the relativeposition of 101 and 102. Furthermore, there can be an existing patternof molecules (110) on the solid support (102). The traveling droplets(106) can be aligned to the existing pattern on the surface. One shouldappreciate that the alignment and the dispensing are crucial steps insome embodiments. Multiple reagents can be dispensed to the sites on thesurface in a sequential process. The solid support (102) is also knownas the destination surface. This surface may have molecules that arepreviously deposited on the surface. These molecules can represent acomplex pattern on the surface of (102). Furthermore, these moleculesmay be covalently bonded, hydrogen bonded, or not bonded (just depositedin solution or dry form) to the surface of (102). In a preferredembodiment, the droplets (106) created by the mechanical wave actuateddispenser (103, 180) are aimed and deposited at desired positions on thesurface of 102. Adjacent surface droplets (111) can be combined by thecreation of “merger” droplets (112) by positioning the merger droplets(112) in between or around the surface droplets (111). In thisembodiment, the alignment of the droplets (106) to the solid support(102) and the molecules attached to the solid support (110) is crucial.A system can be devised to align the droplet to the patterns (molecules)on 102 by using a variety of sensing methods. In some embodiments, theposition of the dispensed droplet in relation to the existing pattern on102 is known or determinable by the user. The detection method can bebased on acoustics, electrical conductive, electrical capacitive, oroptical.

In FIG. 1A, the alignment is illustrated using an optical setup. A setof test droplets (170) can be dispensed to several locations on thesolid support (102). The relative position between the test droplets(170: A surface droplet dispensed for the purpose of alignment of thedroplet to the solid attached molecules (111)) and fixed registrationmarks (172: A surface mark fixed on the solid support (102) to provide areference position on the solid support) on the surface of 102 canprovide information on the alignment between the source plate (101) andthe destination surface (102). A computer system can be used tocalculate a set of correction (offset) parameters, which will be used tocorrect the alignment by adjusting the positioning motors controllingthe position of 101, 102, and 103.

The same mechanical wave actuated dispenser can be used to dispensemiscible or non-miscible solution onto the droplet. Miscible solution(e.g. in water) may contain enzymes, dNTP, buffer, primers or anycombination thereof. One should appreciate that the size of the dropletis determined by the volume and by the surface tension of the solution.One drawback is that the smaller the droplet, the faster it willevaporate. In some embodiments, a non-miscible solution (e.g. oil) canbe used. A non-miscible solution will have the advantage to protect thedroplet from evaporation and from its environment.

Methods for assembling large polynucleotides using oligonucleotidesattached to a solid support are provided herein. In one embodiment,droplets containing polymerase, dNTPs, buffer, chemicals, primers or anycombination thereof are spotted or jetted onto specific features orlocation containing oligonucleotides (or spots) on the solid support. Insome embodiments, the solid surface does not any free hydroxyl group.One should appreciate that in some embodiments that the features oractive or synthesis surface are hydrophilic and the non-active or inertsurface areas (e.g. between features) are hydrophobic. According to someembodiments, a protective coating such as a hydrophilic or hydrophobiccoating (depending upon the nature of the solvent) is utilized overportions of the substrate to be protected, sometimes in combination withmaterials that facilitate wetting by the reactant solution in otherregions. In some embodiments, portion of the solid support compriseshydrophilic sites and portion of the solid support comprises hydrophobicsites. In some embodiments, a hydrophilic site is inert to conditions ofin situ synthesis and assembly. Yet in other embodiments, a hydrophobicsite is inert to conditions of in situ synthesis and assembly. Ahydrophilic site may include free amino, hydroxyl, carboxyl, thiol,amido, halo or sulfonate group as well as modified forms thereof. Ahydrophobic site may include alkyl, fluoro group as well as modifiedforms thereof. The synthesis sites may support covalent or non-covalentattachment of chemicals or biological molecules. For instance, thehydrophilic sites may support attachment of a linker moiety (polylysineetc.). In some embodiments, the synthesis surface area is located at theextremity of the solid support. For example, the solid support may befirst reacted with a suitable reagent to form a hydrophilic surface.Part of the solid support is then treated with a suitable reagent toform hydrophobic surface to allow polymer synthesis. Alternatively, thesolid support may be first reacted with suitable reagent to form ahydrophilic surface. Part of the hydrophilic surface may be protectedwith a suitable reagent to form a hydrophobic surface. The hydrophilicsynthesis surface area may then be deprotected to allow polymersynthesis. Alternatively, the area between hydrophilic synthesissurfaces may be coated with a dewetting agent such as wax or polymers.The dewetting agent may be inkjetted or patterned with other techniques(e.g. lithography). One should appreciate that after dispensing thedroplets onto the active sites, droplets are separated from the othersby surface tension of the solution. In some embodiments, merging ormixing of two adjacent droplets is prevented by the hydrophobic areaaround the active sites. In another embodiment, a dewetting agent isinkjetted or spotted in between the active features to build a verticalheight between the active features thereby forming a micro-well array.For example, wax or polymers can be spotted using a mechanical waveactuated dispenser.

In some embodiments, oligonucleotides or polynucleotides are amplifiedwithin the droplet by solid phase PCR thereby eluting the amplifiedsequences into the droplet. In other embodiments, oligonucleotides orpolynucleotides are first detached form the solid support and thanamplified. In one embodiment, covalently-attached oligonucleotides aretranslated into surface supported DNA molecules through a process ofgaseous cleavage using amine gas. Oligonucleotides can be cleaved withammonia, or other amines, in the gas phase whereby the reagent gas comesinto contact with the oligonucleotide while attached to, or in proximityto, the solid support (see Boal et al., NAR, 1996 (24(15):3115-7), U.S.Pat. Nos. 5,514,789; 5,738,829 and 6,664,388). In this process, thecovalent bond attaching the oligonucleotides to the solid support iscleaved by exposing the solid support to the amine gas under elevatedpressure and/or temperature.

In some embodiments, oligonucleotides or polynucleotides are extendedonce to prepare a complementary strand hybridized to the surface-boundtemplate. A second processing step is performed to melt and remove theamplicons from the templates. This second processing step can beachieved via enzymatic activity (e.g., helicase), buffer condition,temperature, or other methods where hydrogen bonds between complementarystrands could be compromised. The extension followed by melting processcan be performed repeatedly to achieve multiple copies of each template.

In some embodiments, oligonucleotides are synthesized in situ andcomprises a cleavable linker moiety. Oligonucleotides may then becleaved by exposure to conditions such as acid, base, oxidation,reduction, heat, light, metal ion catalysis, displacement or eliminationchemistry or by enzymatic cleavage. Under such conditions,oligonucleotides can be eluted within the droplet without beingamplified.

In one embodiment, oligonucleotides may be attached to a solid supportthrough a cleavable linkage moiety. For example, the solid support maybe functionalized to provide cleavable linkers for covalent attachmentto the oligonucleotides. The linker moiety may be of six or more atomsin length. Alternatively, the cleavable moiety may be within anoligonucleotide and may be introduced during in situ synthesis. A broadvariety of cleavable moieties are available in the art of solid phaseand microarray oligonucleotide synthesis (see e.g., Pon, R., MethodsMol. Biol. 20:465-496 (1993); Verma et al., Annu. Rev. Biochem.67:99-134 (1998); U.S. Pat. Nos. 5,739,386, 5,700,642 and 5,830,655; andU.S. Patent Publication Nos. 2003/0186226 and 2004/0106728). A suitablecleavable moiety may be selected to be compatible with the nature of theprotecting group of the nucleoside bases, the choice of solid support,and/or the mode of reagent delivery, among others. In an exemplaryembodiment, the oligonucleotides cleaved from the solid support containa free 3′-OH end. Alternatively, the free 3′-OH end may also be obtainedby chemical or enzymatic treatment, following the cleavage ofoligonucleotides. The cleavable moiety may be removed under conditionswhich do not degrade the oligonucleotides. Preferably the linker may becleaved using two approaches, either (a) simultaneously under the sameconditions as the deprotection step or (b) subsequently utilizing adifferent condition or reagent for linker cleavage after the completionof the deprotection step.

The covalent immobilization site may either be at the 5′ end of theoligonucleotide or at the 3′ end of the oligonucleotide. In someinstances, the immobilization site may be within the oligonucleotide(i.e. at a site other than the 5′ or 3′ end of the oligonucleotide). Thecleavable site may be located along the oligonucleotide backbone, forexample, a modified 3′-5′ internucleotide linkage in place of one of thephosphodiester groups, such as ribose, dialkoxysilane, phosphorothioate,and phosphoramidate internucleotide linkage. The cleavableoligonucleotide analogs may also include a substituent on, orreplacement of, one of the bases or sugars, such as 7-deazaguanosine,5-methylcytosine, inosine, uridine, and the like.

In one embodiment, cleavable sites contained within the modifiedoligonucleotide may include chemically cleavable groups, such asdialkoxysilane, 3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate,3′-(N)-phosphoramidate, 5′-(N)phosphoramidate, and ribose. Synthesis andcleavage conditions of chemically cleavable oligonucleotides aredescribed in U.S. Pat. Nos. 5,700,642 and 5,830,655. For example,depending upon the choice of cleavable site to be introduced, either afunctionalized nucleoside or a modified nucleoside dimer may be firstprepared, and then selectively introduced into a growing oligonucleotidefragment during the course of oligonucleotide synthesis. Selectivecleavage of the dialkoxysilane may be effected by treatment withfluoride ion. Phosphorothioate internucleotide linkage may beselectively cleaved under mild oxidative conditions. Selective cleavageof the phosphoramidate bond may be carried out under mild acidconditions, such as 80% acetic acid. Selective cleavage of ribose may becarried out by treatment with dilute ammonium hydroxide.

In another embodiment, a non-cleavable hydroxyl linker may be convertedinto a cleavable linker by coupling a special phosphoramidite to thehydroxyl group prior to the phosphoramidite or H-phosphonateoligonucleotide synthesis as described in U.S. Patent ApplicationPublication No. 2003/0186226. The cleavage of the chemicalphosphorylation agent at the completion of the oligonucleotide synthesisyields an oligonucleotide bearing a phosphate group at the 3′ end. The3′-phosphate end may be converted to a 3′ hydroxyl end by a treatmentwith a chemical or an enzyme, such as alkaline phosphatase, which isroutinely carried out by those skilled in the art.

In another embodiment, the cleavable linking moiety may be a TOPS (twooligonucleotides per synthesis) linker (see e.g., PCT publication WO93/20092). For example, the TOPS phosphoramidite may be used to converta non-cleavable hydroxyl group on the solid support to a cleavablelinker. A preferred embodiment of TOPS reagents is the Universal TOP S™phosphoramidite. Conditions for Universal TOPS™ phosphoramiditepreparation, coupling and cleavage are detailed, for example, in Hardyet al, Nucleic Acids Research 22(15):2998-3004 (1994). The UniversalTOPS™ phosphoramidite yields a cyclic 3′ phosphate that may be removedunder basic conditions, such as the extended ammonia and/orammonia/methylamine treatment, resulting in the natural 3′ hydroxyoligonucleotide.

In another embodiment, a cleavable linking moiety may be an aminolinker. The resulting oligonucleotides bound to the linker via aphosphoramidite linkage may be cleaved with 80% acetic acid yielding a3′-phosphorylated oligonucleotide.

In another embodiment, the cleavable linking moiety may be aphotocleavable linker, such as an ortho-nitrobenzyl photocleavablelinker. Synthesis and cleavage conditions of photolabileoligonucleotides on solid supports are described, for example, inVenkatesan et al. J. of Org. Chem. 61:525-529 (1996), Kahl et al., J. ofOrg. Chem. 64:507-510 (1999), Kahl et al., J. of Org. Chem. 63:4870-4871(1998), Greenberg et al., J. of Org. Chem. 59:746-753 (1994), Holmes etal., J. of Org. Chem. 62:2370-2380 (1997), and U.S. Pat. No. 5,739,386.Ortho-nitobenzyl-based linkers, such as hydroxymethyl, hydroxyethyl, andFmoc-aminoethyl carboxylic acid linkers, may also be obtainedcommercially.

In some embodiments, two adjacent droplets containing two multiplecopies of different oligonucleotides or polynucleotides in solution arecombined by merging the appropriate droplets on the solid support asillustrated in FIG. 2 and FIGS. 3A-3D. In FIG. 2, the solid supportcomprises different and unique molecules (201, 202, 203, 204) supportedor attached to the surface of 102, a unique molecule (250) supported orattached to the surface of 102 at multiple positions other uniquemolecules (299) supported or attached to the surface of 102. On thesolid support surface (102) an existing pattern of molecules can befound. Different molecules or oligonucleotides can exist at differentpositions, as shown by the placement of 201, 202, 203, 204, 250, and299. One should appreciate that the arrangement of these uniquemolecules (201, 202, 203, 204) can be designed to strategically allowthe subsequent combining of the contents of these sites. For example,these unique molecules can be arranged in a checker board pattern. Thefirst checker board pattern contains 201 and 202. After individualreactions within the microvolume of 201 and 202 are complete, the usercan choose to combine the content of 201 and 202 by forming a dropletthat encompasses both 201 and 202 sites. In another subsequent step, thecontent of 201+202 can be combined with the content of 203+204, to forma reaction that contains all four reaction products of the uniquemolecules (201, 202, 203, 204). FIG. 3A illustrates the same generalconcept with A, B, C, D. In step 0, all the unique molecules are reactedin separate volumes. In Step 1, the adjacent sites are combined, to giveA+B, and C+D, etc. In Step 2, A+B can be combined with C+D, and etc. InStep 3, another level of aggregation is added. One should appreciatethat there is no limit to the number of steps that can be implemented.In FIG. 3B and FIG. 3C, two possible arrangement strategies areoutlined. In the first strategy, some adjacent sites comprise the samemolecules or oligonucleotides (e.g. A and B) and the four sites may becombined to generate a circular droplet (FIG. 3B). In the secondstrategy, each site comprises a unique and different molecule oroligonucleotide (FIG. 3C).

For example, with reference to FIG. 2, solid supported oligonucleotide201 and oligonucleotides 202 may be amplified in first stage droplet 1and first stage droplet 2. After amplification, each first stage dropletcontains one amplified double stranded oligonucleotide sequence. Inembodiments, multiple copies of oligonucleotides 201 and multiple copiesof oligonucleotide 202 are eluted within the first stage droplet 1 andthe first stage droplet 2, respectively. The two first stage dropletsbeing in close proximity to each other are combined to form a secondstage droplet. In some embodiments, two different or moreoligonucleotides or polynucleotides may be immobilized or synthesized atthe same location (or feature) on the solid support thereby facilitatingtheir interaction after amplification within the same droplet. See e.g.US 2004/0101894. In some embodiments, droplets are merged to form biggerdroplets by adding, or spotting additional “merger” droplets in betweenor around the appropriate original droplets. The additional “merger”droplets may contain enzyme (e.g. polymerase, ligase, etc.), additionaloligonucleotides and all reagents to allow assembly by PCR or byligation (enzymatic or chemical) or by any combination of enzymaticreaction. For example, oligonucleotides in a given droplet may hybridizeto each other and may assemble by PCR or ligation. The bigger dropletsor second stage droplets contain polynucleotides subassemblies and canbe subsequently merged to form larger droplets or third stage dropletcontaining larger fragments. As used herein the term subassembly refersto a nucleic acid molecule that has been assembled from a set ofoligonucleotides. Preferably, a subassembly is at least 2-fold or morelong than the oligonucleotides. For example, a subassembly may be about100, 200, 300, 400, 500, 600, or ore bases long. One should appreciatethat the use of droplets as isolated reaction volume enables a highlyparallel system. In some embodiments, at least 100, at least 1,000reactions can take place in parallel. In some embodiments, the primersare immobilized on the substrate in close proximity to the spotscontaining the oligonucleotides to be assembled. In some embodiments,the primers are cleaved in situ. In some embodiments, the primers aresupported on the solid support. The primers may then be cleaved in situand eluted within a droplet that will subsequently merged with a dropletcontaining solid supported or eluted oligonucleotides.

In some embodiments, mechanical wave actuated delivery techniques (e.g.,a surface acoustic wave device, a piezoelectric device, or other forcetransducer) can be used to transfer droplets from a first spot orposition to a second spot or position on a solid support, forminghopping droplets. A hopping droplet can include one or moreoligonucleotides, enzymes, buffers, dNTPs, etc. One or more ofdenaturing, restriction, annealing, ligation, and chain extension can becarried out in the hopping droplet. The first and second spots can betwo neighboring spots on a solid support or separated by any distance.The two spots can each have a feature. In one embodiment, the featuresat the two spots share the same or complementary sequence at oneterminus. The hopping droplet technique can be combined with travelingdroplet for the assembly of a target nucleic acid product orintermediate.

FIG. 3D is a schematic representation of an exemplary assembly strategyof hierarchical gene synthesis. Each circle represents a spot or adroplet on a solid support. As depicted in FIG. 3D, seven successivechain extension and/or ligation reactions are conducted to assemblesixty-four oligonucleotides 262 in parallel to ultimately produce afinal desired assembly 274. Specifically, upon completion of synthesisof the sixty-four individual oligonucleotides 262, these are thenpair-wise assembled to thirty-two subassemblies 264, which are thenassembled into subassemblies 266, 268, 270, 272, until the finalfull-length product 274 is synthesized.

In some embodiments, methods to control temperature on-chip so thatenzymatic reactions can take place on chip (PCR, ligation or any othertemperature sensitive reaction) are provided. In some embodiments, ascanning laser is used to control the thermocycling on distinct spots onthe solid support. The wavelength used can be chosen from wide spectrum(100 nm to 100,000 nm, i.e. from ultraviolet to infrared). In someembodiments, the feature on which the droplet is spotted comprises anoptical absorber or indicator. In some other embodiment, opticalabsorbent material can be added on the surface of the droplet. In someembodiments, a dye can be added to the droplet reaction volume. In someembodiments, the solid support is cooled by circulation of air or fluid.The energy to be deposited can be calculated based on the absorbancebehavior. In some embodiments, the temperature of the droplet can bemodeled using thermodynamics. The temperature can be measured by an LCDlike material or any other in-situ technology. Yet in anotherembodiment, the whole substrate can be heated and cooled down to allowenzymatic reactions to take place.

One method to control the temperature of the surface droplets is byusing a scanning optical energy deposition setup is shown in FIG. 4. Anenergy source (406) can be directed by a scanning setup (407) to depositenergy at various locations on the surface of the solid support (401)comprising attached or supported molecules (404). Optical absorbentmaterial (402, 405) can be added on the surface of the solid support oron the surface of droplet. Optical energy source, such as a highintensity lamp, laser, or other electromagnetic energy source (includingmicrowave) can be used. The temperature of the different reaction sites(420, 421, 422, 423, . . . ) can be controlled independently bycontrolling the energy deposited at each of the sites.

For example, a Digital Micromirror Device (DMD) can be used fortemperature control. DMD is an microfabricated spatial opticalmodulator. See, for example, U.S. Pat. No. 7,498,176. In someembodiments, a DMD can be used to precisely heat selected spots ordroplets on the solid support. The DMD can be a chip having on itssurface, for example, several hundred thousand to several millionmicroscopic mirrors arranged in a rectangular array which correspond tothe spots or droplets to be heated. The mirrors can be individuallyrotated (e.g., ±10-12°), to an on or off state. In the on state, lightfrom a light source (e.g., a bulb) is reflected onto the solid supportto heat the selected spots or droplets. In the off state, the light isdirected elsewhere (e.g., onto a heatsink). In one example, the DMD canconsist of a 1024×768 array of 16 μm wide micromirrors. In anotherexample, the DMD can consist of a 1920×1080 array of 10 μm widemicromirrors. Other arrangements of array sizes and micromirror widthsare also possible. These mirrors can be individually addressable and canbe used to create any given pattern or arrangement in heating differentspots on the solid support. The spots can also be heated to differenttemperatures, e.g., by providing different wavelength for individualspots, and/or controlling time of irradiation.

Aspects of the methods and devices provided herein relate to determiningthe sequence of one or more polynucleotide and/or selectively isolatingthe polynucleotides having the correct sequences of interest. In someembodiments, methods to sequence verify assembled polynucleotides usinghigh throughput sequencing are provided. In some embodiments, theassembled polynucleotides are assembled on a different station. In someembodiments, assembled polynucleotides are sequenced by synthesis.Sequence determinations can be made by any available method permittingthe querying of the sequence of an individual molecule (“single moleculesequencing”), whether directly or through the querying of an amplifiedpopulation of nucleic acids derived from a single molecule (“polonysequencing”). Generally, the method of sequence determination should benon-destructive, to the extent that the objective of the sequencedetermination is the identification of a subsequently usefuloligonucleotide.

Methods of polymerase amplification and sequencing are described, forexample, in U.S. Patent Application Nos. 2005-0079510 and 2006-0040297;in Mitra et al., (2003) Analytical Biochemistry 320: 55-65; Shendure etal., (2005) Science 309:1728-1732; and in Margulies et al., (2005)Nature 437:376-380, the complete disclosures of each of which are hereinincorporated by reference. As discussed in Shendure et al., (2005)Science 309:1729, polony amplification can involve, for example, in situpolonies, in situ rolling circle amplification, bridge PCR, picotiterPCR, or emulsion PCR. Generally, an oligonucleotide to be amplified isprepared to include primer binding sites, whether as part of itssequence when initially synthesized or by subsequent ligation to adaptormolecules bearing the primer binding sites.

Prior to sequencing, the oligonucleotides are immobilized at distinctlocations (e.g., predetermined, addressable locations or randomlocations) on a solid support. In the Genome Sequencer 20 System from454 Life Sciences, for example, beads from polony amplification aredeposited into wells of a fiber-optic slide. In the method of Shendureet al., beads from polony amplification are poured in a 5% acrylamidegel onto a glass coverslip manipulated to form a circular gelapproximately 30 microns thick, giving a disordered monolayer.

The oligonucleotides that have been immobilized on a solid support, canthen be sequenced by any non-destructive method such as sequencing bysynthesis, permit the iterative interrogation of nucleobases of anoligonucleotide, which is advantageous when iterative interrogationprovides a higher accuracy determination of sequence identity. Forexample, Margulies et al., describe a sequencing by synthesis techniquein which the polony beads are sequenced in picoliter-sized wells using apyrosequencing protocol. As another approach, Shendure et al., describea four color sequencing by ligation method in which the identity ofnucleobases is iteratively determined by ligation of anchor primers tosecond primers. The second primers are labeled with fluorescent dyes,the color of which identifies the nucleobase at one position in theprimer; other positions are degenerate. Because ligation occurs onlywhen the anchor primer and the second primer are properly annealed, thecolor of the second primer identifies the nucleobase in theoligonucleotide at the position corresponding to the non-degenerateposition of the second primer. By stripping the complexes and repeatingthe process with different populations of second primers in which thenon-degenerate position varies, nucleobases in the oligonucleotide canbe iteratively identified. In yet another approach, Mitra et al.,describe methods for sequencing polonies in parallel by fluorescent insitu sequential quantitation, “FISSEQ”; i.e., by performing repeatedcycles of primer extension with reversibly-labeled fluorescentdeoxynucleotides (for example, cycling sequentially through dATP, dCTP,dGTP, and dUTP or dTTP,). Incorporation of labeled dNTPs is monitoredusing a scanning fluorescence microscope and software for automatedimage alignment and sequence calling. If a polony has incorporated abase, it will fluoresce, thereby identifying the template baseimmediately 3′ of the primer. Once the incorporated base is identified,the dye linker is cleaved by a reducing agent (for example, by thiolreduction), or exposure to near UV light. Cleaved dye is washed away andthe cycle is repeated by adding a different dye-labeled base, washingaway unincorporated dNTP, and scanning the gel. The sequence of thetemplate nucleic acid is compiled as the primed template is interrogatedat each cycle for incorporated nucleotide.

The technology provided herein can embrace any method of non-destructivesequencing. Non-limiting examples of non-destructive sequencing includepyrosequencing, as originally described by Hyman et al., (1988, AnalBiochem 74: 324-436) and bead-based sequencing, described for instanceby Leamon et al., (2004, Electrophoresis 24: 3769 3777). Non-destructivesequencing also includes methods using cleavable labeledoligonucleotides, as the above described Mitra et al., (2003, AnalBiochem 320:55-62) and photocleavable linkers (Seo et al., 2005, PNAS102: 5926-5933). Methods using reversible terminators are also embracedby the technology provided herein (Metzker et al., 1994, NAR 22:4259-4267). Further methods for non-destructive sequencing (includingsingle molecule sequencing) are described in U.S. Pat. No. 7,133,782 andU.S. Pat. No. 7,169,560 which are hereby incorporated by reference.

Methods to selectively extract or isolate the correct sequence from theincorrect sequences are provided herein. The term “selective isolation”,as used herein, can involve physical isolation of a desiredpolynucleotide from others as by selective physical movement of thedesires polynucleotide; selective inactivation, destruction, release, orremoval of other polynucleotide than the polynucleotide of interest. Itshould be appreciated that a polynucleotide or library ofpolynucleotides assembled according to methods provided herein mayinclude some errors that may result from sequence errors introducedduring the oligonucleotides synthesis, the synthesis of the assemblynucleic acids and/or from assembly errors during the assembly reaction.Unwanted nucleic acids may be present in some embodiments. For example,between 0% and 50% (e.g., less than 45%, less than 40%, less than 35%,less than 30%, less than 25%, less than 20%, less than 15%, less than10%, less than 5% or less than 1%) of the sequences in a library may beunwanted sequences. In one embodiment, all polynucleotide constructs aresequenced in a sequencing channel. In some embodiments, thepolynucleotide constructs can be sequenced in situ on the solid supportused in gene synthesis and reused/recycled therefrom. Analysis of thesequence information from the oligonucleotides permits theidentification of those polynucleotides that appear to have desirablesequences and those that do not. Such analysis of the sequenceinformation can be qualitative, e.g., providing a positive or negativeanswer with regard to the presence of one or more sequences of interest(e.g., in stretches of 10 to 120 nucleotides). In some embodiments,polynucleotides of interest can then be selectively isolated from therest of the population. The sorting of individual polynucleotides can befacilitated by the use of one or more solid supports (e.g. bead,insoluble polymeric material, planar surface, etc. . . . ) to which thepolynucleotides are attached. Polynucleotides determined to have thecorrect desired sequence can be selectively released or selectivelycopied.

If the polynucleotides are located in different locations e.g. inseparate wells of a substrate, polynucleotides can be taken selectivelyfrom the wells identified as containing polynucleotides with desirablesequences. For example, in the apparatus of Margulies et al., polonybeads are located in individual wells of a fiber-optic slide. Physicalextraction of the bead from the appropriate well of the apparatuspermits the subsequent amplification or purification of the desirablepolynucleotides free of other contaminating polynucleotides.Alternatively, if the polynucleotides are attached to the beads using aselectively cleavable linker, cleavage of the linker (e.g., byincreasing the pH in the well to cleave a base-labile linker) followedby extraction of the solvent in the well can be used to selectivelyisolate the polynucleotides without physical manipulation of the bead.Likewise, if the method of Shendure et al., is used, physical extractionof the beads or of the portions of the gel containing thepolynucleotides of interest can be used to selectively isolate desiredpolynucleotides.

Certain other methods of selective isolation involve the targeting ofpolynucleotide molecules without a requirement for physical manipulationof a solid support. Some such methods incorporate the use of an opticalsystem to specifically target radiation to individual polynucleotidemolecules. In one embodiment, destructive radiation is selectivelytargeted against undesired polynucleotides (e.g., using micromirrortechnology) to destroy or disable them, leaving a population ofoligonucleotides enriched for desired polynucleotides. This enrichedpopulation can then be released from solid support and/or amplified,e.g., by PCR.

Example of methods and systems for selectively isolating the desiredproduct (e.g. polynucleotide of interest) are shown in FIGS. 5A-5E. Inone embodiment, the system comprises a microfluidics reaction chamber(701) for sequencing reaction; individual sequencing reaction sites,containing undesirable material (702), reaction site containing desiredpopulation (703), an outlet (exit) (799) of the chamber (FIG. 5A).

In one embodiment, the correct sequence polonie is trapped using a lasertweezer or optical tweezer (FIGS. 5B-5C). Laser tweezers have been usedfor approximately two decades in the fields of biotechnology, medicineand molecular biology to position and manipulate micrometer-sized andsubmicrometer-sized particles (A. Ashkin, Science, (210), pp 1081-1088,1980). By focusing the laser beam on the desired vessel (e.g. bead,etc.) comprising the desired polynucleotide of interest, the desiredvessel remain optically trapped in the sequencing channel while theundesired polynucleotide sequences are eluted. One embodiment of themethod and device is illustrated in FIG. 5B wherein the system furthercomprises a laser and lens system (750) to implement optical tweezers.In order to retain the vessel(s) which contains the desirableproduct(s), an optical tweezer setup (750) can be used. Objects at thefocal point (751) of the optical tweezer can be retained while the restof the material is washed off (towards the exit 799). Once all of theundesirable materials are washed off, the optical tweezer can be tunedoff allowing the release the desired population or polynucleotide.

Another method to capture the desirable products is by ablating theundesirable products. In one embodiment, a high power laser is used togenerate enough energy to disable, degrade, or destroy the product orproducts (e.g. polynucleotides) in areas where undesirable materialsexist (763). The area where desirable products) exist (764) does notreceive any destructive energy, hence preserving its contents.

In yet another implementation, the desirable product can be detected andtracked by using a camera and vision system (FIG. 5D). The vision system(775) will follow the position of the desirable product (or products),until it reaches the sorting junction (777). Desirable material will bedirected towards a collection channel (774) while undesirable product(or products) directed towards another (773), where the sortingmechanism is controlled by two or more flow controllers (776).

In yet another implementation, a method to achieve “selective isolation”can involve the utilization of photopolymers to retain and trapdesirable products (FIG. 5E). For example, commercial sequencingplatforms, for example, those developed by PACIFIC BIOSCIENCES™,VISIGEN® Biotechnologies, Inc., and other companies utilize a surfacebond polymerase in a process termed sequencing-by-synthesis. In suchprocesses, a template (in some cases a circularized template) iscaptured by the surface attached polymerase during sequencing. Thetemplate remains captured after sequencing is complete, and this allowsfor a “selective isolation” method to be applied to this class of nextgeneration sequencing platforms. In one embodiment, after sequencing iscomplete (FIG. 5E(1)), and the amplicons are removed via washing (FIG.5E(2)), a photo-sensitive polymer precursor can be introduced to thesurface that encapsulates the areas of interest (FIG. 5E(3)). The soldsupport can be, for example, a glass substrate and a polymerase can beimmobilized on the solid support. The wells can be wells in a layer, forexample, a metal layer on the solid support. In FIG. 5E(1), a templatestrand can be associated with the polymerase, and the amplicons can besynthesized from the template using the polymerase and labelednucleotides in solution. A pattern of radiation (controlled by scanninglaser, a DMD, or other optical manipulation) is introduced to allowpolymerization of the photo-sensitive polymer to take place on thelocations where product capture is desirable. The unexposedphoto-sensitive polymer precursors are removed in a wash, leaving theundesirable products exposed to the fluid environment, allowing exposureto buffers, enzymes, and chemical to facilitate the removal of theseproducts (FIG. 5E(4)). Subsequently, the polymerized polymers can beremoved, exposing the desired products (FIG. 5E(5)). These products canbe eluted from the surface for collection, or amplified in situ with thesurface-attached polymerase to produce an enriched volume of the finalproduct (FIG. 5E(6)).

In some embodiments, assembled library nucleic acids may be amplified,sequenced or cloned. In some embodiments, a host cell may be transformedwith the assembled library nucleic acids. Library nucleic acids may beintegrated into the genome of the host cell. In some embodiments, thelibrary nucleic acids may be expressed, for example, under the controlof a promoter (e.g., an inducible promoter). Individual variant clonesmay be isolated from a library. Nucleic acids and/or polypeptides ofinterest may be isolated or purified. A cell preparation transformedwith a nucleic acid library, or an isolated nucleic acid of interest,may be stored, shipped, and/or propagated (e.g., grown in culture).

Aspects of the methods and devices provided herein may includeautomating one or more acts described herein. In some embodiments, oneor more steps of an assembly reaction may be automated using one or moreautomated sample handling devices (e.g., one or more automated liquid orfluid handling devices). Automated devices and procedures may be used todeliver reaction reagents, including one or more of the following:starting nucleic acids, buffers, enzymes (e.g., one or more ligases,polymerases, nucleases, helicases, and/or other enzymes), nucleotides,salts, and any other suitable agents such as stabilizing agents.Automated devices and procedures also may be used to control thereaction conditions. For example, an automated thermal cycler may beused to control reaction temperatures and any temperature cycles thatmay be used. In some embodiments, a scanning laser may be automated toprovide one or more reaction temperatures or temperature cycles suitablefor incubating polynucleotides. Similarly, subsequent analysis ofassembled polynucleotide products may be automated. For example,sequencing may be automated using a sequencing device and automatedsequencing protocols. Additional steps (e.g., amplification, cloning,etc.) also may be automated using one or more appropriate devices andrelated protocols. It should be appreciated that one or more of thedevice or device components described herein may be combined in a system(e.g., a robotic system) or in a micro-environment (e.g., amicro-fluidic reaction chamber). Assembly reaction mixtures (e.g.,liquid reaction samples) may be transferred from one component of thesystem to another using automated devices and procedures (e.g., roboticmanipulation and/or transfer of samples and/or sample containers,including automated pipetting devices, micro-systems, etc.). The systemand any components thereof may be controlled by a control system.

In some embodiments, a droplet can be dried (e.g., via evaporation ofthe solvent) and/or heated to an elevated temperature to achieve enzymedeactivation after a desired/predetermined enzymatic reaction step. Suchenzyme deactivation provides methods for enzyme control in addition tothe other methods disclosed herein (e.g., washing, chemical, bufferconditions, temperature control of the droplet, and the like).

After dry deactivation, the surface location (e.g., where the evaporateddroplet was located) can be rehydrated (e.g., to re suspend themolecules that had become temporarily surface-supported). In this way,the functionality of molecules that are not affected by the dry down andheat treatment can be preserved while the functionality of moleculesthat are affected by the dry down and/or heating can be selectivelydeactivated and/or destroyed.

Accordingly, method steps and/or aspects of the devices provided hereinmay be automated using, for example, a computer system (e.g., a computercontrolled system). A computer system on which aspects of the technologyprovided herein can be implemented may include a computer for any typeof processing (e.g., sequence analysis and/or automated device controlas described herein). However, it should be appreciated that certainprocessing steps may be provided by one or more of the automated devicesthat are part of the assembly system. In some embodiments, a computersystem may include two or more computers. For example, one computer maybe coupled, via a network, to a second computer. One computer mayperform sequence analysis. The second computer may control one or moreof the automated synthesis and assembly devices in the system. In otheraspects, additional computers may be included in the network to controlone or more of the analysis or processing acts. Each computer mayinclude a memory and processor. The computers can take any form, as theaspects of the technology provided herein are not limited to beingimplemented on any particular computer platform. Similarly, the networkcan take any form, including a private network or a public network(e.g., the Internet). Display devices can be associated with one or moreof the devices and computers. Alternatively, or in addition, a displaydevice may be located at a remote site and connected for displaying theoutput of an analysis in accordance with the technology provided herein.Connections between the different components of the system may be viawire, optical fiber, wireless transmission, satellite transmission, anyother suitable transmission, or any combination of two or more of theabove.

Each of the different aspects, embodiments, or acts of the technologyprovided herein can be independently automated and implemented in any ofnumerous ways. For example, each aspect, embodiment, or act can beindependently implemented using hardware, software or a combinationthereof. When implemented in software, the software code can be executedon any suitable processor or collection of processors, whether providedin a single computer or distributed among multiple computers. It shouldbe appreciated that any component or collection of components thatperform the functions described above can be generically considered asone or more controllers that control the above-discussed functions. Theone or more controllers can be implemented in numerous ways, such aswith dedicated hardware, or with general purpose hardware (e.g., one ormore processors) that is programmed using microcode or software toperform the functions recited above.

In this respect, it should be appreciated that one implementation of theembodiments of the technology provided herein comprises at least onecomputer-readable medium (e.g., a computer memory, a floppy disk, acompact disk, a tape, etc.) encoded with a computer program (i.e., aplurality of instructions), which, when executed on a processor,performs one or more of the above-discussed functions of the technologyprovided herein. The computer-readable medium can be transportable suchthat the program stored thereon can be loaded onto any computer systemresource to implement one or more functions of the technology providedherein. In addition, it should be appreciated that the reference to acomputer program which, when executed, performs the above-discussedfunctions, is not limited to an application program running on a hostcomputer. Rather, the term computer program is used herein in a genericsense to reference any type of computer code (e.g., software ormicrocode) that can be employed to program a processor to implement theabove-discussed aspects of the technology provided herein.

It should be appreciated that in accordance with several embodiments ofthe technology provided herein wherein processes are stored in acomputer readable medium, the computer implemented processes may, duringthe course of their execution, receive input manually (e.g., from auser).

Accordingly, overall system-level control of the assembly devices orcomponents described herein may be performed by a system controllerwhich may provide control signals to the associated nucleic acidsynthesizers, liquid handling devices, thermal cyclers, sequencingdevices, associated robotic components, as well as other suitablesystems for performing the desired input/output or other controlfunctions. Thus, the system controller along with any device controllerstogether form a controller that controls the operation of a nucleic acidassembly system. The controller may include a general purpose dataprocessing system, which can be a general purpose computer, or networkof general purpose computers, and other associated devices, includingcommunications devices, modems, and/or other circuitry or components toperform the desired input/output or other functions. The controller canalso be implemented, at least in part, as a single special purposeintegrated circuit (e.g., ASIC) or an array of ASICs, each having a mainor central processor section for overall, system-level control, andseparate sections dedicated to performing various different specificcomputations, functions and other processes under the control of thecentral processor section. The controller can also be implemented usinga plurality of separate dedicated programmable integrated or otherelectronic circuits or devices, e.g., hard wired electronic or logiccircuits such as discrete element circuits or programmable logicdevices. The controller can also include any other components ordevices, such as user input/output devices (monitors, displays,printers, a keyboard, a user pointing device, touch screen, or otheruser interface, etc.), data storage devices, drive motors, linkages,valve controllers, robotic devices, vacuum and other pumps, pressuresensors, detectors, power supplies, pulse sources, communication devicesor other electronic circuitry or components, and so on. The controlleralso may control operation of other portions of a system, such asautomated client order processing, quality control, packaging, shipping,billing, etc., to perform other suitable functions known in the art butnot described in detail herein.

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

EXAMPLES Example 1: Utilize Solid-Attached Molecular Library

A solid support with a high density of high diversity DNA moleculesattached is used as the destination surface. The DNA molecules on thesurface are arranged in a pattern that corresponds to a known map storedin a computer. On the surface, several alignment features are placednext to the DNA molecule pattern, preferably one at each of the fourcorners of the DNA pattern. The placement of the alignment feature isknown in relation to the DNA pattern.

First, as illustrated in FIG. 1A and FIG. 1B, a set of droplets (106,and 170) is deposited next to the alignment features. The positionaldifference between these droplets and the alignment features is measuredwith a microscope objective and camera. The positional difference iscompared to the desired positions of the droplets, and the offset of thealignments is calculated. This set of calculated values are stored andused to compensate for the alignment between the source plate and thedestination surface.

In the source plate, the reagent wells (105) contain reagents toamplify, digest, and assemble DNA molecules. For example, first, a DNApolymerase, appropriate primers, salts, and dNTPs are dispensed to asite on the surface of the solid support containing a template sequenceattached to the said surface. After the dispensing, the temperature ofthe surface is modulated to induce polymerase chain reaction (PCR). Theresult is that the template sequences are amplified into solution in thedroplet.

After amplification, a polymerase disabling agent (for example epoxyATP) is added to the reaction volume. A few cycles of PCR reaction takesplace, and disables the polymerase enzymes.

The primer sites on the amplification product in solution are removed byusing a restriction enzyme that cuts outside of its recognition frame.The restriction enzyme is added to the reaction site, and allowed todigest the substrate to produce only the construction pieces. Therestriction enzymes are heat in-activated.

At this point, the double stranded material in each of the dropletcontains, among other molecules, hybridized oligonucleotides suitablefor subsequent assembly. In a massively parallel reaction, all of thereaction sites on the solid support surface can be treated in parallel.For example, in a case where there are 8 fragments are assembledtogether in a 3 step process, A, B, C, D, E, F, G, H are all processedsimultaneously in 8 individual reaction volumes.

In the next step, as illustrated in FIG. 3A, two reaction volumes arecombined, i.e., A and B, C and D, E and F, G and H, forming 4 step onereactions. During the combining of these droplets, DNA ligase is addedto the reaction volumes, and the samples are treated to allow ligationreaction to complete. Once this step (step one) is complete, these fourlarger droplets are further aggregated into two even larger droplets,for example, A+B is combined with C+D, and E+F is combined with G+H.More reagent is added during the combination process, and allowed toligate under suitable conditions. Finally, the two large droplets arecombined into a final droplet, with the addition of reagents forsubsequent reactions, such as DNA ligase. The final volume is allowed toreact at the optimal condition.

Example 2: Utilize Molecular Library without Surface-Bond Amplification

A solid support with a high density of high diversity DNA moleculesattached is used as the destination surface. The DNA molecules on thesurface are arranged in a pattern that corresponds to a known map storedin a computer. On the surface, several alignment features are placednext to the DNA molecule pattern, preferably one at each of the fourcorners of the DNA pattern. The placement of the alignment feature isknown in relation to the DNA pattern.

First, as illustrated in FIGS. 1A and 1B, a set of droplets (106, and170) is deposited next to the alignment features. The positionaldifference between these droplets and the alignment features is measuredwith a microscope objective and camera. The positional difference iscompared to the desired positions of the droplets, and the offset of thealignments is calculated. This set of calculated values are stored andused to compensate for the alignment between the source plate and thedestination surface.

Instead of adding reagents that will amplify the surface attached DNAmolecules, a reagent is added which can cleave the DNA molecules fromthe surface. After cleaving, these DNA molecules can participate insubsequent reactions in a similar manner as outlined in the previousexample.

Example 3: Cleaving without Using Liquid Reagents

In another example, the strategy outlined in Example 2 can be simplifiedto not use liquid cleaving reagents. Surface attached (covalent) DNAmolecules can be translated into surface supported DNA molecules througha process of gaseous cleavage using amine gas. In this process, thesolid support with surface attached molecules is exposed to a amine gasunder elevated pressure and/or temperature, this process is shown to beuseful to cleave the covalent bond that attaches the DNA molecules tothe solid support. Subsequent steps after this gaseous cleavage isidentical to those of example 1 and 2.

The resulting surface-supported high density molecular array library canbe accessed (e.g., to the resolution of individual spots), for example,by the various droplet-based technologies disclosed herein. Inparticular, the application of gaseous cleavage to high-densitymolecular libraries can offer a useful and unique advantage. During thegaseous cleavage of a high-density library, the elements of themolecular library can be transformed from being surface-attached tosurface-supported. Therefore, the information of the location of each ofthe elements is preserved, allowing for subsequent manipulation and/oraccess to the surface-supported molecules. In conjunction with thetechniques disclosed herein, the application of gaseous cleavage canallows for each and every element on a high-density molecular library tobe individually accessed.

Cleaved oligos can be use directly to participate in the assemblyreaction, or can be amplified via PCR prior to the assembly reactions.

Example 4: Using a Scanning Laser for Thermal Cycling

After dispensing PCR reagents onto the solid support (as in Example 1),a scanning laser is used for thermocycling. The temperature of theindividual sites can be modulated to perform PCR reaction with differentthermal parameters (temperature profile) at each of the sites. Forexample, depending on sequence information, site A may need a differentoptimal temperature profile compared to site B, and it is possible toaccommodate such temperature differences. Also, mixed reaction can takeplace on the same solid support, i.e., site C requires a PCR reactionwhile at the same time, site D requires a ligation or digestionreaction. In such a case, the temperature profile at site C can be onefor a PCR reaction while the temperature at site D can be maintained ata steady temperature for ligation or digestion.

Example 5: Commercial Microarray-Based Assembly

The solid support can be a DNA microarray (e.g., a chip). The microarraycan be constructed, custom ordered or purchased from a commercial vendor(e.g., Agilent, Affymetrix, Nimblegen). Commercially available DNAmicroarrays generally have an arrayed series of microscopic spots of DNAoligonucleotides, each containing picomoles of a specific DNA sequence(e.g., 20 to 120 nucleotides). The spots combined can represent agenomic or subgenomic feature (e.g., a human chromosome, the Drosophilagenome, the yeast genome, etc.). Thus commercial DNA microarrays can beused as source of oligonucleotides for gene and/or genome assembly. Someof the advantages include: commercial microarrays have high density anddiversity of source materials, and can be washed and reused multipletimes; cost per base of raw material can be reduced by a factor of 10 to100, 100 to 1,000, or 1,000 to 10,000 compared to other methods (e.g.,de novo synthesis); and high diversity allows for higher synthesisoutput by parallel processing. Thus repeatable, reliable, automated DNAassembly can be achieved using commercial microarrays.

An example of commercial microarray is the Agilent DNA microarrays.These microarrays can have 1×1M, 1×244K, 2×105K, 4×44K, or 8×15K spotsone a single slide, and about 10⁵ to about 10¹⁵ molecules per spot. Thespot size and spacing between neighboring spots can each be less thanabout 100 microns. In various examples, the spot size can be less thanabout 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25,20, 15, or 10 microns. In various examples, the spacing between spotscan be less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45,40, 35, 30, 25, 20, 15, or 10 microns. Spot sizes and spacing can varyindependently and can be uniform or non-uniform across a microarray. Adatabase of 28 million+predesigned, in si/ico-validated aCGH probes thatspan exonic, intronic, and intergenic DNA regions can be deposited onthese microarrays.

1. On-Chip Oligonucleotide Synthesis

Oligonucleotides can be synthesized on a microarray using the attachedoligonucleotides as template. For example, oligonucleotide synthesis canbe achieved within a droplet covering each spot by solid phase PCR. Thedroplet can contain enzymes, buffers, dNTPs, primers, etc. In somecases, the PCR primers are attached to a solid surface (Adessi et al.,Nucleic Acids Research, 2000, Vol. 28, No. 20 e87). Another techniquehinges on the attachment of the template to a surface (Bennett S.,Pharmacogenomics, 2004 June; 5(4):433-8).

DNA microarrays can have very high density of oligonucleotides on thesurface, which can generate steric hindrance to polymerases needed forPCR. Theoretically, the oligonucleotides are generally spaced apart byabout 2 nm to about 6 nm. For polymerases, a typical 6-subunit enzymecan have a diameter of about 12 nm. Therefore the microarray needs to becustom treated to address the surface density issue such that thespacing of surface-attached oligonucleotides can accommodate thephysical dimension of the enzyme. For example, a subset of theoligonucleotides can be chemically or enzymatically cleaved, orphysically removed from the microarray. Other methods can also be usedto modify the oligoucleotides such that when primers are applied andannealed to the oligonucleotides, at least some 3′ hydroxyl groups ofthe primers (start of DNA synthesis) are accessible by polymerase. Thenumber of accessible 3′ hydroxyl groups per spot can be stochastic orfixed. For example, the primers, once annealed, can be treated to removesome active 3′ hydroxyl groups, leaving a stochastic number of 3′hydroxyl groups that can be subject to chain extension reactions. Inanother example, a large linker molecule (e.g., a concatamer) can beused such that one and only one start of synthesis is available perspot, or in a subset of the oligonucleotides per spot.

In some embodiments, a dendron can be used for modification ofmicroarray surface. A dendron is a molecular structure that resembles atree. See FIG. 6. By branching out away from the surface of themicroarray, a dendron can reduce the steric hindrance effects. Thedendron can also be produced in a way that some or all of the branchesare chemically inert, with exception to one or more reactive groups(e.g., for the attachment of one or more oligonucleotides, resulting inone or more 3′ end as the start of DNA synthesis per dendron). Doing socan reduce the surface density of reactive groups, leading to a lessdense DNA microarray.

In some embodiments, the modification of the surface-attached moleculardensity can be modulated by chemical cleaving after completing thesynthesis of the molecular library. A cleaving site can be designed intothe synthesis process to allow the harvesting of the synthesizedproducts. For example, amine can be used for cleaving oligonucleotidesfrom the surface. Such post-synthesis cleaving step can be applied toreduce the surface molecular density. In one implementation, thecleaving process can be allowed to proceed only for a short period oftime or under other non-optimal reaction conditions to achievein-complete cleavage of the synthesized product. The remaining (e.g.,uncleaved) product that remains attached to the surface will have areduced surface molecular density, which can facilitate subsequentenzymatic or chemical processing.

Microarray features are in general too small for conventional fluidhandling technologies. Pico-liter and sub pico-liter volume droplets canbe used to access the large library of material available on a DNAmicroarray. See FIGS. 7A-7B. This access is possible because thedimension of the droplets matches that of the features on themicroarray. Pico-liter and sub pico-liter droplet based liquid handlingand manipulation can be achieved using e.g., the inkjet technology. Toperform chemistry at the scale of the microarray, if assuming a surfaceliquid-solid contact angle of 90 degrees, the volumes for each spot isbetween about 0.3 pL to about 2 nL. These volumes coincides with inkjetdroplet technology well. For example, a typical inkjet printer iscapable of producing 1.5 to 10 pL droplets, while other commercialultrasonic dispensing techniques can produce droplets down to 0.6 pL.

A droplet 810, as illustrated in FIG. 8, can be dispensed (e.g., inkjetted) on a microarray 800. The droplet 810 can contain variousreagents such as enzymes, buffers, dNTPs, primers, etc. The droplet 810covers a spot 820 (a feature corresponding to a predefined sequence) onthe microarray 800. For purpose of illustration only, fouroligonucleotides, 801, 802, 803, 804 are shown, while many moreoligonucleotides having the same sequence are also present on spot 820but not shown. PCR can be carried out to synthesize oligonucleotides801′, 802′, 803′, 804′ complementary to template oligonucleotides 801,802, 803, 804 that are attached to spot 820.

Any DNA polymerase having a chain extension activity can be suitable forthe on-chip oligonucleotide synthesis. In one embodiment, stranddisplacing polymerase exo-(having no exonuclease activity) can be usedfor isothermal nucleic acid amplification. The polymerase can be used toextend once or more than once (e.g., multiple times) per templateoligonucleotide. For example, the polymerase can bind a template andextend, fall off the end of the template, and bind to the same oranother surface template and extend again. This leads to a linear (notexponential) amplification. The resulted linearly amplified pool ofproducts are single-stranded DNA (ssDNA).

Primers can be included in the reaction mixture for linear and/orexponential amplification. The primers can have the same sequence ordifferent sequences. In one embodiment, a pair of primers, one sensestrand and the other anti-sense strand, can be added to the reactionmixture such that amplification is exponential and yieldsdouble-stranded DNA (dsDNA). The primer sequences attached to the PCRproducts (e.g., ssDNA or dsDNA) can be cleaved by using a Uracil DNAglycosylase. The cleaved products can be digested into single, double,triple, or other short nucleotide sequences by using a) the same UracilDNA glycosylase; and/or b) a primer design that has the followingpattern: UXUXU . . . XUXU (where X is any base) or UXnUXnUXn . . . XnU(where Xn is a series of X numbered n, e.g., ATG can be represented byX3). In the case of Xn, n can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, or more. In another example, the primer sequence is in the patternof UnXnUnXn . . . XnUnXn, where Un is n repeats of U (e.g., U3 is UUU).

After the PCR reaction, the polymerase can be deactivated to preventinterference with subsequent steps. A heating step (e.g., hightemperatures) can denature and deactivate most enzymes but not likely toaffect thermally stable PCR DNA polymerase. A non-thermal stable versionof polymerase can be used in PCR, which are generally less optimized inerror rate and speed. Alternatively, adenosine riboepoxide triphosphatenucleotide (epoxy dATP) can be used to inactivate polymerase. Epoxy dATPcan be incorporated into a lengthening DNA polymer and form a covalentbond to the enzyme during incorporation, hence block the active site ofthe enzyme. Another method is to remove or wash away all reagents,including the polymerase, leaving only the duplexes formed between thetemplate oligonucleotides and synthesized oligonucleotides on themicroarray surface. For example, liquids can evaporate in a vacuum whileheating. Enzymes can be heat deactivated at the same time, with orwithout liquid. Heat deactivation of enzymes in the absence of liquidcan be advantageous without hurting (e.g., hydrolysis) otherbiologically active molecules (e.g., DNA molecules). After deactivation,a washing step can be used to remove deactivated enzymes and othernon-surface bound molecules. Next, the microarray surface (e.g.,location of drop foot print) can be re-activated by adding a solvent(e.g., water) to the surface.

In some embodiments, spots on the microarray can containoligonucleotides that are substantially complementary (e.g., 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100%). As a result, the PCR productssynthesized from such two spots are also substantially complementary andcan form duplexes when combined (e.g., by merging two droplets). Theseduplexes can be subjected to error and/or mismatch recognition andremoval (see below; e.g., using a mismatch recognition protein such asMutS), where mismatch-free duplexes can be exponentially amplifiedthrough additional rounds of PCR.

2. Error Control

With reference to FIG. 8, template oligonucleotides 801, 802, 803, 804can have inherent errors as they are generally chemically synthesized(e.g., deletions at a rate of 1 in 100 bases and mismatches andinsertions at about 1 in 400 bases). Assuming an average error rate of 1in 300 bases and an average template oligonucleotide size of 70 bases,every 1 in 4 template oligonucleotides will contain an error compared toa reference sequence (e.g., the wide-type sequence of a gene ofinterest). For example, template oligonucleotide 803 can contain anerror 805 which can be a mismatch, deletion, or insertion. In PCRsynthesis, the error 805 is retained in the synthesized oligonucleotide803′ as error 805′. Additional errors (not shown) can be introducedduring PCR. Methods for error correction and/or filtration are neededfor high-fidelity gene synthesis/assembly.

In one embodiment, error-containing oligonucleotides are removed by amethod illustrated in FIG. 8. PCR products (duplexes 801-801′, 802-802′,803-803′, 804-804′) are denatured by melting the duplexes (e.g., atelevated temperature, using a helicase, etc.), forming freeoligonucleotides 801′, 802′, 803′, 804′. In some embodiments, using ahelicase to melt duplexes can provide for isothermal denaturing withoutelevating the temperature.

Next, under annealing conditions (e.g., lower temperature),oligonucleotides 801′, 802′, 803′, 804′ will randomly anneal to templateoligonucleotides 801, 802, 803, 804. By way of example, new duplexes801-803′, 802-801′, 803-804′ and 804-802′ can be formed. 802-801′ and804-802′ are error-free duplexes, whereas 801-803′ and 803-804′ eachcontain a mismatch between the two complementary strands. All duplexeswithin a spot are then subject to a stringent melting step to denature801-803′ and 803-804′, leaving 802-801′ and 804-802′ intact.Oligonucleotides 803′ (containing error 805′) and 804′ can then beremoved or washed away. Error-free oligonucleotides 801′ and 802′ can bemelted and recovered in a droplet for subsequent amplification,ligation, and/or chain extension. These steps can be repeated multipletimes to enrich for error-free oligonucleotides, as microarray 800 canbe washed and reused at least several times.

The conditions for stringent melt (e.g., a precise melting temperature)can be determined by observing a real-time melt curve. In an exemplarymelt curve analysis, PCR products are slowly heated in the presence ofdouble-stranded DNA (dsDNA) specific fluorescent dyes (e.g., SYBR Green,LCGreen, SYTO9 or EvaGreen). With increasing temperature the dsDNAdenatures (melts), releasing the fluorescent dye with a resultantdecrease in the fluorescent signal. The temperature at which dsDNA meltsis determined by factors such as nucleotide sequence, length and GC/ATratio. Melt curve analysis can detect a single base difference. Variousmethods for accurate temperature control at individual spots can be usedas disclosed above.

Another embodiment of the invention is directed toward the recognitionand removal of double-stranded oligonucleotides containing sequencemismatch errors. It is particularly related to the removal oferror-containing oligonucleotides generated, for example, by chemical orbiological synthesis by removing mismatched duplexes using mismatchrecognition proteins (MMBP). For methods and materials known in the artrelated to error detection and correction using mismatch bindingproteins, see e.g., International Publication No. WO 03/054232, and U.S.Patent Publication Nos. 20050227235 and 20060127926; incorporated byreference in their entirety. For example, MutS error filtration can beused to remove error-containing oligonucleotides, in which abulge-binding protein is used to remove mismatch-containing DNAdouble-strands. In some embodiments, all double-strands can bechemically or enzymetically cleaved off the microarry. The protein-DNAcomplex can be captured by an affinity of the MMBP protein to, e.g., aspecific antibody, immobilized nickel ions (e.g., where MMBP is producedas a his-tag fusion), streptavidin (e.g., where MMBP has been modifiedby the covalent addition of biotin) or by any other such mechanisms asare common to the art of protein purification. These affinity-basedcapture methods can be assisted by acoustic, magnetic, and/or opticalseparation methods. Alternatively, the protein-DNA complex is separatedfrom the pool of error-free DNA sequences by a difference in mobility,such as by size-exclusion column chromatography or by electrophoresis.

Error filtration using MMBP proteins can also be achieved withoutcleaving the PCR products off the microarray. For example, the presenceof the protein-DNA complex can change helicase activity in meltingdouble-strands; under suitable conditions (e.g., low temperature, shortreaction time) only mismatch-free double-strands that are not bound byMMBP proteins are melted thereby releasing error-free synthesizedoligonucleotides. In some embodiments, the MMBP protein (e.g., MutS) canbe irreversibly complexed with a mismatch recognition protein by theaction of a chemical crosslinking agent (e.g., dimethyl suberimidate,DMS), or of another protein (such as MutL). This blocks access toerror-containing oligonucleotides attached on the microarry insubsequent steps (e.g., additional rounds of PCR). The population ofdouble strands that are error free can then be subjected to one or moreof: denaturing, chain extension, annealing, restriction, and ligation.

3. Hierarchical Assembly

Step-wise hierarchical assembly (e.g., as discussed above) can be usedto construct polynucleotides. Neighboring droplets, each containing aunique sequence, can be manipulated (e.g., moved, merged) to aggregateand assemble the content of individual droplets in a way that minimizesthe complexity in biochemistry, hence improving assembly efficiency. Adroplet can be moved from one oligonucleotide-containing spot to anotheroligonucleotide-containing spot, to allow assembly (e.g., ligationbased, chain extension based) of the two different oligonucleotides. Twoneighboring droplets can be merged by dispensing liquid (e.g., asolution containing for example, buffer, dNTPs, and enzymes that allowligation and/or chain extension) in between. Two neighboring dropletscan also be moved to an oligonucleotide-free position where pair-wiseassembly can be performed without any oligonucleotide annealing back tothe oligonucleotides attached on the microarray.

Mechanical wave actuated delivery techniques (e.g., a surface acousticwave device, a piezoelectric inkjet device) can be used to move or hopdroplets. More advanced droplet manipulations, such as moving andsplitting droplets can be facilitated by using electrostatic or magneticforce (Cho et. al., Journal of Microelectromechanical Systems, 12(1),2003, pp. 70-80).

To facilitate automation, the single-step polymerase assemblymulti-plexing (PAM) reaction developed by Tian et al. (Nature 432,1050-1054 (23 Dec. 2004)) can be used for multiple gene syntheses from acombined/merged pool of oligonucleotides. For PAM, gene-flanking primerpairs can be added to the pool of oligonucleotides (with the primerpairs at a higher concentration than the oligonucleotides), togetherwith thermostable polymerase and dNTPs. Extension of overlappingoligonucleotides and subsequent amplification of multiple full-lengthgenes can thus be accomplished in a one-step reaction. Different genericadaptor sequences can be incorporated into the ends of each gene or geneset, and a set of complementary adaptor-primer pairs can bepre-synthesized to avoid the cost of synthesizing gene-specific PAMprimer pairs and to facilitate automation.

4. Sequence Verification

After assembly, all products can be pooled and all impurities (e.g.,enzymes, dNTPs, primers) can be removed. The assembled products can be acomplex pool of fragments containing correct and incorrect assemblies.This pool of fragments can be sequenced and the desired products havingthe correct sequences can be recovered. A preparative sequencing stepcan be performed to find and recover the correct sequences from amongthe mixed population of DNA molecules. A 2nd or 3rd generation DNAsequencer (e.g. a polonator) or, for example, a system such as a PACIFICBIOSCIENCE™ SMRT™ system can be used, e.g., for single moleculesequencing, as discussed above.

In some embodiments, qualitative sequencing can also meet the goal ofverifying the correct sequence without employing a sequencer. Amicroarray having substantially the same features as the microarraypreviously used in oligonucleotide synthesis and assembly can providesuch qualitative sequencing information. For example, the samemicroarray previously used in oligonucleotide synthesis and assembly canbe washed, dried, and reused, thereby reducing costs. Alternatively, anew microarray having substantially the same features can be used; aftersequencing, this microarray can be washed, dried, and reused forsynthesis, assembly, and/or sequencing.

In qualitative sequencing, the assembled products can hybridize tooligonucleotides immobilized on the microarray under hybridizationconditions (e.g., proper temperature, buffer), where a positivehybridization signal within a particular feature indicates the presenceof the sequence corresponding to that particular feature. For example,the hybridization buffer can contain double-stranded DNA (dsDNA)specific fluorescent dyes (e.g., SYBR Green, LCGreen, SYTO9 orEvaGreen); thus presence or absence of fluorescence at differentfeatures represents positive or negative hybridization, respectively.These individual features on the microarray can thus be used assequencing patches that can provide qualitative information with regardto the sequence of the assembled products.

In various embodiments, the sequencing step can be massively parallel tomatch the output from the assembly process. For example, multiplemicrofluidics chips can be operated simultaneously to achieve highthroughput. In one embodiment, 2, 4 or even 8 microfluidics chips(cores) can be operated in the same sequencing machine at the same time,to allow up to 8 genes to be assembled during a single run. Where amicroarray is used, as many genes can be assembled at once as the amountof DNA material is available on a single chip. Multiple microarrays canalso be used at the same time to further increase parallelism.

Example 6: Synthesis of Oligonucleotides Having a Predefined Sequence

A plurality of oligonucleotides having a predefined sequence aresynthesized by providing a plurality of support-bound templateoligonucleotides in a solution comprising a primer, a polymerase andnucleotides, wherein each of the plurality of template oligonucleotidescomprises a predefined sequence and includes a primer binding site, andwherein the primer comprises at least one nuclease recognition site. Theplurality of template oligonucleotides are exposed to conditionssuitable for primer hybridization and polymerase extension, therebyextending the primers to produce a complementary oligonucleotide foreach of the plurality of template oligonucleotides. The hybridizedcomplementary oligonuceotides are released from the templateoligonucleotides into solution and exposed to a nuclease underconditions suitable for the nuclease to bind to the nuclease recognitionsite on the primer and cleave the primer from complementaryoligonucleotide. The complementary and template oligonucleotides areexposed to conditions suitable for hybridization; thereby to produce aplurality of partially double-stranded oligonucleotides. The pluralityof partially double-stranded oligonucleotides are washed and then thecomplementary oligonucleotides are melted from the templateoligonucleotides.

According to another version, a microarray of single stranded DNAtemplate molecules is provided. Second, a primer, dNTP, and a polymeraseis dispensed (e.g., ink jetted) onto the microarray in spots. The primerincludes at least one, and preferably more than one, nuclease site.Next, a product is produced by priming the template and extending anoligonucleotide on the template. Then, the product is melted apart fromthe template. The product includes the primer sequence as well as thepayload sequence. Subsequently, a nuclease is dispensed (e.g., inkjetted) into the spots. If the primer includes only one nuclease site,it is preferably located between the primer sequence and the payloadsequence, so that the nuclease cleaves the primer from the payload. Whenthe primer includes more than one nuclease site, they are preferablylocated between the primer sequence and the payload sequence as well asbeing dispersed within the primer, so that the nuclease cleaves theprimer from the payload in addition to cleaving the primer into smalleroligonucleotides. After cleavage, the payload is rehybridized onto thesingle stranded DNA template. Due to differences in length and thusmelting temperature, the payload can hybridize at a temperature wherethe cleaved primer remains in solution. After rehybridization, theunbound primer and/or primer fragments are removed by washing. After theprimer and/or primer fragments (and, in some examples other unwantedelements, for example, nuclease) are removed, the payload can be meltedapart from the template and is available is in a substantially puresolution.

EQUIVALENTS

The present invention provides among other things novel methods andapparatuses for high-fidelity gene assembly. While specific embodimentsof the subject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

INCORPORATION BY REFERENCE

Reference is made to PCT Publication Nos. WO07136736 and WO08024319, andU.S. Pat. No. 6,248,521. All publications, patents and sequence databaseentries mentioned herein are hereby incorporated by reference in theirentirety as if each individual publication or patent was specificallyand individually indicated to be incorporated by reference.

1.-83. (canceled)
 84. A method for assembling a polynucleotide having apredefined sequence, the method comprising: (a) providing a plurality ofsupport-bound template oligonucleotides at different features on a solidsupport, wherein each of the plurality of template oligonucleotidescomprises a predefined sequence, a primer binding site and a sequenceregion that is complementary to a sequence region of anotheroligonucleotide present at a different feature; (b) exposing theplurality of template oligonucleotides to conditions suitable for primerextension in presence of a primer, thereby extending the primer toproduce a plurality of complementary oligonucleotides; (c) releasing theplurality of complementary oligonucleotides having complementarysequence regions in a droplet; and (d) exposing the plurality ofcomplementary oligonucleotides to conditions suitable for hybridization,ligation, or polymerase extension to assemble the polynucleotide havingthe predefined sequence.
 85. The method of claim 84, wherein the step ofexposing is within a droplet comprising the primer, a polymerase,nucleotides and solvent and wherein the method further comprisesevaporating the solvent.
 86. The method of claim 84, wherein in the stepof releasing, the oligonucleotides are suspended in a droplet.
 87. Themethod of claim 84, wherein the plurality of template oligonucleotidesare synthesized in situ onto a support surface.
 88. The method of claim84, wherein the plurality of template oligonucleotides are immobilizedonto a support surface.
 89. A method for removing error-containingoligonucleotides synthesized on a solid support, the method comprising:(a) synthesizing a first plurality of oligonucleotides in a chainextension reaction, wherein a second plurality of oligonucleotidesimmobilized on the solid support serves as templates in the chainextension reaction; (b) denaturing products of the chain extensionreaction; (c) contacting the first plurality of oligonucleotides withthe second plurality of oligonucleotides under hybridization conditionsto form duplexes; and (d) separating error-containing oligonucleotidesfrom oligonucleotides with error-free sequences using a component whichactively selects for a sequence error.
 90. The method of claim 89,wherein the sequence error selecting component comprises a mismatchrecognition protein MutS or a functional homolog of MutS.